Reducing systemic regulatory T cell levels or activity for treatment of a retinal degeneration disorder

ABSTRACT

The present specification discloses a pharmaceutical composition comprising an active agent that causes reduction of the level of systemic immunosuppression in an individual for use in treating a disease, disorder, condition or injury of the CNS. The pharmaceutical composition is administered by a dosage regimen comprising at least one course of therapy, each course of therapy comprising in sequence a treatment session followed by an interval session of non-treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This divisional claims the benefit of priority and filing date pursuantto 35 U.S.C. § 121 to U.S. Non-Provisional patent application Ser. No.15/261,945, filed Sep. 10, 2016, an application that claims the benefitof priority and the filing date under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/279,622, filed Jan. 15, 2016, U.S.Provisional Patent Application No. 62/353,549, filed Jun. 22, 2016, andU.S. Provisional Patent Application No. 62/358,530, filed Jul. 5, 2016;and U.S. Non-Provisional patent application Ser. No. 15/261,945, filedSep. 10, 2016, is a Continuation-in-Part that claims the benefit ofpriority and the filing date under 35 U.S.C. § 120 to InternationalPatent Application No. PCT/IL2016/050750, filed Jul. 13, 2016; and U.S.Non-Provisional patent application Ser. No. 15/261,945, filed Sep. 10,2016, is a Continuation-in-Part that claims the benefit of priority andthe filing date under 35 U.S.C. § 120 U.S. patent application Ser. No.14/850,794, filed Sep. 10, 2015, a continuation-in-part that claimspriority to U.S. patent application Ser. No. 14/797,894, filed on Jul.13, 2015, a Continuation-in-Part that claims priority to InternationalPatent Application No. PCT/IL2015/050265, filed Mar. 12, 2015, in whichthe United States is designated, and claims the benefit of priority fromU.S. Provisional Patent Application No. 61/951,783, filed Mar. 12, 2014,and U.S. Provisional Patent Application No. 62/030,164, filed Jul. 29,2014, the entire content of each of which is hereby incorporated byreference in its entirety as if fully disclosed herein.

FIELD

The present invention relates in general to methods and compositions fortreating disease, disorder, condition or injury of the Central NervousSystem (CNS) by transiently reducing the level of systemicimmunosuppression in the circulation.

BACKGROUND

Most central nervous system (CNS) pathologies share a commonneuroinflammatory component, which is part of disease progression, andcontributes to disease escalation. Among these pathologies isAlzheimer's disease (AD), an age-related neurodegenerative diseasecharacterized by progressive loss of memory and cognitive functions, inwhich accumulation of amyloid-beta (A13) peptide aggregates wassuggested to play a key role in the inflammatory cascade within the CNS,eventually leading to neuronal damage and tissue destruction (Akiyama etal, 2000; Hardy & Selkoe, 2002; Vom Berg et al, 2012). Despite thechronic neuroinflammatory response in neurodegenerative diseases,clinical and pre-clinical studies over the past decade, investigatingimmunosuppression-based therapies in neurodegenerative diseases, haveraised the question as to why anti-inflammatory drugs fall short(Breitner et al, 2009; Group et al, 2007; Wyss-Coray & Rogers, 2012). Weprovide a novel answer that overcomes the drawbacks of existingtherapies of AD and similar diseases and injuries of the CNS; thismethod is based on our unique understanding of the role of the differentcomponents of systemic and central immune system in CNS maintenance andrepair.

SUMMARY

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an active agent that causes reduction of thelevel of systemic immunosuppression in an individual for use in treatinga disease, disorder, condition or injury of the CNS that does notinclude the autoimmune neuroinflammatory disease, relapsing-remittingmultiple sclerosis (RRMS), wherein said pharmaceutical composition isfor administration by a dosage regimen comprising at least two coursesof therapy, each course of therapy comprising in sequence a treatmentsession followed by an interval session of non-treatment.

In another aspect, the present invention provides a method for treatinga disease, disorder, condition or injury of the Central Nervous System(CNS) that does not include the autoimmune neuroinflammatory diseaserelapsing-remitting multiple sclerosis (RRMS), said method comprisingadministering to an individual in need thereof a pharmaceuticalcomposition comprising an active agent that causes reduction of thelevel of systemic immunosuppression according to the present invention,wherein said pharmaceutical composition is administered by a dosageregime comprising at least two courses of therapy, each course oftherapy comprising in sequence a treatment session followed by aninterval session of a non-treatment period.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B depict the choroid plexus (CP) activity along diseaseprogression in the 5XFAD transgenic mouse model of AD (AD-Tg). (FIG. 1A)mRNA expression levels for the genes icam1, vcam1, cxcl10 and ccl2,measured by RT-qPCR, in CPs isolated from 1, 2, 4 and 8-month old AD-Tgmice, shown as fold-change compared to age-matched WT controls (n=6-8per group; Student's t test for each time point). (FIG. 1B)Representative microscopic images of CPs of 8-month old AD-Tg mice andage-matched VVT controls, immunostained for the epithelial tightjunction molecule Claudin-1, Hoechst nuclear staining, and the integrinlignad, ICAM-1 (scale bar, 50 μm). In all panels, error bars representmean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 2A-C show (FIG. 2A) Quantification of ICAM-1 immunoreactivity inhuman postmortem CP of young and aged non-CNS diseased, and AD patients(n=5 per group; one-way ANOVA followed by Newman-Keuls post hocanalysis); (FIG. 2B) flow cytometry analysis for IFN-γ-expressing immunecells (intracellularly stained, and pre-gated on CD45) in CPs of 8-monthold AD-Tg mice and age-matched WT controls. Shaded histogram representsisotype control (n=4-6 per group; Student's t test); and (FIG. 2C) mRNAexpression levels of ifn-γ, measured by RT-qPCR, in CP tissues isolatedfrom 4- and 8-month old AD-Tg mice, compared to age-matched WT controls(n=5-8 per group; Student's t test for each time point). In all panels,error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 3A-B depict (FIG. 3A) representative flow cytometry plots of CD4⁺Foxp3⁺ splenocyte frequencies (pre-gated on TCRβ) in 8-month old AD-Tgand VVT control mice; and (FIG. 3B) quantitative analysis of splenocytesfrom 1, 2, 4 and 8-month AD-Tg and WT control mice (n=6-8 per group;Student's t test for each time point). In all panels, error barsrepresent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIG. 4 shows gating strategy and representative flow cytometry plots ofsplenocytes from AD-Tg/Foxp3-DTR^(+/−) mice, 1 day after the lastinjection of DTx. DTx was injected i.p. for 4 constitutive days,achieving ˜99% depletion of Foxp3⁺ cells.

FIGS. 5A-G show the effects of transient depletion of Tregs in AD-Tgmice. (FIG. 5A) AD-Tg/Foxp3-DTR^(+/−) (which express the DTR transgene)and a non-DTR-expressing AD-Tg littermate (AD-Tg/Foxp3-DTR⁻) controlgroup were treated with DTx for 4 constitutive days. CP mRNA expressionlevels for the genes icam1, cxcl10 and ccl2, measured by RT-qPCR, in6-month old DTx-treated AD-Tg mice, day after the last DTx injection(n=6-8 per group; Student's t test). (FIGS. 5B-D) Flow cytometryanalysis of the brain parenchyma (excluding the choroid plexus, whichwas separately excised) of 6-month old DTx-treated AD-Tg mice andcontrols, 3 weeks following the last DTx injection. Quantitative flowcytometry analysis showing increased numbers of CD11b^(high)/CD45^(high)mo-Mϕ and CD4⁺ T cells (FIG. 5B), and representative flow cytometryplots (FIG. 5C) and quantitative analysis (FIG. 5D) of CD4⁺Foxp3⁺ Tregfrequencies, in the brain parenchyma of AD-Tg/Foxp3-DTR⁺ mice andAD-Tg/Foxp3-DTR⁻ controls treated with DTx (n=3-7 per group; Student's ttest). (FIG. 5E) mRNA expression levels of foxp3 and il10 in the brainparenchyma of 6-month old DTx-treated AD-Tg AD-Tg/Foxp3-DTR⁺ andAD-Tg/Foxp3-DTR-contros, 3 weeks after the last DTx injection (n=6-8 pergroup; Student's t test). (FIG. 5F) quantitative analysis of GFAPimmunostaining, showing reduced astrogliosis in hippocampal sectionsfrom 6-month old DTx-treated AD-Tg/Foxp3-DTR⁺ and AD-Tg/Foxp3-DTR⁻control mice, 3 weeks following the last DTx injection (scale bar, 50μm; n=3-5 per group; Student's t test). (FIG. 5G) mRNA expression levelsof il-12p40 and tnf-a in the brain parenchyma, 3 weeks following thelast DTx infection (n=6-8 per group; Student's t test). In all panels,error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 6A-E show the effect of transient depletion of Tregs on Aβ plaqueslearning/memory performance. (FIG. 6A) Representative microscopic imagesand (FIG. 6B) quantitative analysis of the brains of 5-month oldDTx-treated AD-Tg/Foxp3-DTR⁺ and AD-Tg/Foxp3-DTR⁻ control mice, 3 weeksafter the last DTx injection, immunostained for Aβ plaques and Hoechstnuclear staining (scale bar, 250 μm). Mean Aβ plaque area and numbers inthe hippocampal dentate gyrus (DG) and the 5^(th) layer of the cerebralcortex were quantified (in 6 μm brain slices; n=5-6 per group; Student'st test). FIGS. 6C-E) show Morris water maze (MWM) test performance of6-month old DTx-treated AD-Tg/Foxp3-DTR⁺ and control mice, 3 weeks afterthe last DTx injection. Following transient Treg depletion, AD-Tg miceshowed better spatial learning/memory performance in the (FIG. 6C)acquisition, (FIG. 6D) probe and (FIG. 6E) reversal phases of the MWM,relative to AD-Tg controls (n=7-9 per group; two-way repeated measuresANOVA followed by Bonferroni post-hoc analysis for individual paircomparisons; *, P<0.05 for overall acquisition, probe, and reversal). Inall panels, error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***,P<0.001.

FIG. 7 shows mRNA expression levels of ifn-γ, measured by RT-qPCR, inCPs isolated from 6- and 12-month old APP/PS1 AD-Tg mice (a mouse modelfor Alzheimer's disease (see Materials and Methods)), compared toage-matched VVT controls (n=5-8 per group; Student's t test). Error barsrepresent mean±s.e.m.; *, P<0.05.

FIGS. 8A-I show the therapeutic effect of administration of weeklyGlatiramer acetate (GA) in AD-Tg mice. (FIG. 8A) Schematicrepresentation of weekly-GA treatment regimen. Mice (5-month old) weres.c. injected with GA (100 μg), twice during the first week (on day 1and 4), and once every week thereafter, for an overall period of 4weeks. The mice were examined for cognitive performance, 1 week (MWM), 1month (RAWM) and 2 months (RAWM, using different experimental spatialsettings) after the last injection, and for hippocampal inflammation.FIGS. 8B-D show mRNA expression levels of genes in the hippocampus ofuntreated AD-Tg mice, and AD-Tg mice treated with weekly-GA, at the ageof 6 m, showing (FIG. 8B) reduced expression of pro-inflammatorycytokines such as TNF-α, IL-1β and IL-12p40, (FIG. 8C) elevation of theanti-inflammatory cytokines IL-10 and TGF-β, and of (FIG. 8D) theneurotropic factors, IGF-1 and BDNF, in weekly-GA treated mice (n=6-8per group; Student's t test). In FIGS. 8E-G, AD-Tg mice (5 months old)were treated with either weekly-GA or with vehicle (PBS), and comparedto age-matched WT littermates in the MWM task at the age of 6 m. Treatedmice showed better spatial learning/memory performance in theacquisition (FIG. 8E), probe (FIG. 8F) and reversal (FIG. 8G) phases ofthe MWM, relative to controls (n=6-9 per group; two-way repeatedmeasures ANOVA followed by Bonferroni post-hoc for individual paircomparisons; VVT mice, black circles; AD-Tg controls, white circles;treated AD-Tg, grey circles). FIGS. 8H-I show cognitive performance ofthe same mice in the RAWM task, 1 month (FIG. 8H) or 2 months (FIG. 8I)following the last GA injection (n=6-9 per group; two-way repeatedmeasures ANOVA followed by Bonferroni post-hoc for individual paircomparisons). Data are representative of at least three independentexperiments. In all panels, error bars represent mean±s.e.m.; *, P<0.05;**, P<0.01;***, P<0.001.

FIGS. 9A-H show further therapeutic effects of administration ofweekly-GA in AD-Tg mice. FIGS. 9A-B shows 5XFAD AD-Tg mice that weretreated with either weekly-GA, or vehicle (PBS), and were examined atthe end of the 1^(st) week of the administration regimen (after a totalof two GA injections). Flow cytometry analysis for CD4⁺Foxp3⁺ splenocytefrequencies (FIG. 9A), and CP IFN-γ-expressing immune cells (FIG. 9B;intracellularly stained and pre-gated on CD45), in treated 6-month oldAD-Tg mice, compared to age-matched VVT controls (n=4-6 per group;one-way ANOVA followed by Newman-Keuls post hoc analysis). (FIG. 9C)mRNA expression levels for the genes icam1, cxcl10 and ccl2, measured byRT-qPCR, in CPs of 4-month old AD-Tg mice, treated with either weekly-GAor vehicle, and examined either at the end of the 1^(st) or 4^(th) weekof the weekly-GA regimen (n=6-8 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis). FIGS. 9D-E show representative imagesof brain sections from 6-month old AD-Tg/CX₃CR1^(GFP/+) BM chimerasfollowing weekly-GA. CX₃CR1^(GFP) cells were localized at the CP of thethird ventricle (3V; i), the adjacent ventricular spaces (ii), and theCP of the lateral ventricles (LV; HO in AD-Tg mice treated withweekly-GA (FIG. 9D; scale bar, 25 μm). Representative orthogonalprojections of confocal z-axis stacks, showing co-localization of GFP⁺cells with the myeloid marker, CD68, in the CP of 7-month oldAD-Tg/CX₃CR1^(GFP/+) mice treated with weekly-GA, but not in controlPBS-treated AD-Tg/CX₃CR1^(GFP/+) mice (FIG. 9E; scale bar, 25 μm). (FIG.9F) CX₃CR1^(GFP) cells are co-localized with the myeloid marker IBA-1 inbrains of GA-treated AD-Tg/CX₃CR1^(GFP/+) mice in the vicinity ofAβplaques, and co-expressing the myeloid marker, IBA-1 (scale bar, 25μm). FIGS. 9G-H show representative flow cytometry plots of cellsisolated from the hippocampus of 4-month old VVT, untreated AD-Tg, andAD-Tg mice, on the 2^(nd) week of the weekly-GA regimen.CD11b^(high)/CD45^(high) mo-Mϕ were gated (FIG. 9G) and quantified (FIG.9H; n=4-5 per group; one-way ANOVA followed by Newman-Keuls post hocanalysis). In all panels, error bars represent mean±s.e.m.; *, P<0.05;**, P<0.01;***, P<0.001.

FIGS. 10A-H depict the therapeutic effect of administration of a p300inhibitor (C646) in AD-Tg mice. In FIGS. 10A-B, aged mice (18 months)were treated with either p300i or vehicle (DMSO) for a period of 1 week,and examined a day after cessation of treatment. Representative flowcytometry plots showing elevation in the frequencies of CD4⁺ T cellsexpressing IFN-γ in the spleen (FIG. 10A), and IFN-γ-expressing immunecell numbers in the CP (FIG. 10B), following p300i treatment. FIGS.10C-E show representative microscopic images (FIG. 10C), andquantitative analysis, of Aβ plaque burden in the brains of 10-month oldAD-Tg mice, which received either p300i or vehicle (DMSO) for a periodof 1 week, and were subsequently examined after 3 additional weeks.Brains were immunostained for AR plaques and by Hoechst nuclear staining(n=5 per group; Scale bar, 250 μm). Mean Aβ plaque area and plaquenumbers were quantified in the hippocampal DG (FIG. 10D) and the 5^(th)layer of the cerebral cortex (FIG. 10E) (in 6 μm brain slices; n=5-6 pergroup; Student's t test). (FIG. 10F) Schematic representation of thep300i treatment (or DMSO as vehicle) administration regimen to thedifferent groups of AD-Tg mice at the age of 7 months, in either 1 or 2sessions. FIGS. 10G-H show the change mean of Aβ plaque (5^(th)percentage coverage of the cerebral cortex (5^(th) layer) (FIG. 10G),and the change in mean cerebral soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ protein levels(FIG. 10H), relative to the untreated AD-Tg group (Aβ₁₋₄₀ and Aβ₁₋₄₂mean level in untreated group, 90.5±11.2 and 63.8±6.8 pg/mg totalportion, respectively; n=5-6 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis). In all panels, error bars representmean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 11 A-D show that PD-1 blockade augments percentage ofIFN-γ-producing CD4+ T-cells in the spleen, as well as IFN-γ expressionat the choroid plexus in AD-Tg mice. 10-month old AD-Tg mice were i.p.injected on day 1 and day 4 with 250 ug of either anti-PD-1 or controlIgG, and examined at days 7-10 for the effect on the systemic immuneresponse and CP activity. (FIGS. 11A-B) Representative flow cytometryplots (FIG. 11A), and quantitative analysis (FIG. 11B), of CD4⁺IFN-γ⁺splenocyte frequencies (intracellularly stained and pre-gated on CD45and TCR-β), in anti-PD-1 or IgG treated AD-Tg mice, and untreated AD-Tgand VVT controls (n=4-6 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis; **, P<0.01 between the indicted treatedgroups; error bars represent mean±s.e.m.). (FIG. 11C) mRNA expressionlevels of ifn-g, measured by RT-qPCR in the CP of AD-Tg mice treatedwith anti-PD-1 when compared to IgG treated and untreated AD-Tg controls(FIG. 11D) GO annotation terms enriched in RNA-Seq in CPs of the samemice (n=3-5 per group; one-way ANOVA followed by Newman-Keuls post hocanalysis; *, P<0.05) (gray scale corresponds to negative log-base 10 ofP-value).

FIGS. 12A-B show that PD-1 blockade mitigates cognitive decline in AD-Tgmice. 10-month old AD-Tg mice were i.p. injected on day 1 and day 4 with250 ug of either anti-PD-1 or control IgG, and examined 1 or 2 monthslater for the effect on pathology with (FIG. 12A) showing performance ofAD-Tg mice in the RAWM after 1 treatment session with anti-PD-1 or IgGcontrol and (FIG. 12B) showing effect of single anti-PD-1 treatmentsession, or 2 sessions with a 1 month interval on performance. Singlearrows indicate time points of treatment, and double arrows indicatetime points of cognitive testing. Cognitive performance of anti-PD-1 andIgG treated mice, compared to age-matched VVT and untreated AD-Tg mice,assessed by the average number of errors per day in the RAWM learningand memory task (n=6-8 per group; two-way repeated measures ANOVAfollowed by Bonferroni post-hoc for individual pair comparisons).

FIGS. 13A-D depict representative microscopic images showing that PD-1blockade mitigates AD pathology (FIG. 13A), and quantitative analyses(FIG. 13B, FIG. 13C, FIG. 13D), of Aβ plaque burden and astrogliosis inthe brains of AD-Tg mice, which were treated at the age of 10-monthswith either anti-PD-1 (in 1 or 2 sessions, as depicted in FIG. 12A-B) orIgG control, and subsequently examined at the age of 12 months. Brainswere immunostained for Aβ plaques (in red), GFAP (marking astrogliosis,in green), and by Hoechst nuclear staining (n=4-5 per group; Scale bar,50 μm). Mean Aβ plaque area and plaque numbers were quantified in thehippocampal dentate gyrus (DG) and the 5th layer of the cerebral cortex,and GFAP immunoreactivity was measured in the hippocampus (in 6 μm brainslices; n=5-6 per group; Student's t test). In all panels, error barsrepresent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIG. 14 shows the effect of different dosing and frequency ofadministration of anti-PD-1 antibody on cognitive decline in AD-Tg miceand illustrates the dosage scheme and the effect of anti-PD-1 antibodytreatment on spatial learning and memory performance using the radialarm water maze (RAWM) task at 7 months of age. Black arrows indicatetime points of treatment, and illustrations indicate time points ofcognitive testing.

FIGS. 15A-C show the effect of repeated administration of anti-PD-1antibody on cognitive decline in AD-Tg mice with (FIG. 15A) showingdosage scheme of a single injection followed by 4 weeks of non-treatmentinterval; (FIG. 15B) showing the effect of anti-PD-1 antibody treatmenton spatial learning and memory performance using the radial arm watermaze (RAWM) task at 5 months of age; and (FIG. 15C) showing anti-PD-1antibody effect on spatial learning and memory performance using theradial arm water maze (RAWM) task at 6 months of age.

FIG. 16 shows the effect of a single administration of anti-TIM-3antibody on cognitive decline in AD-Tg mice and illustrates the dosagescheme and the effect of anti-TIM-3 antibody treatment on spatiallearning and memory performance using the radial arm water maze (RAWM)task at 7 months of age. Black arrows indicate time points of treatment,and illustrations indicate time points of cognitive testing.

FIGS. 17A-B PD-L1 blockade mitigates cognitive decline in AD-Tg micewith (FIG. 17A) showing the effect of a single administration ofanti-PD-L1 antibody on cognitive decline in AD-Tg mice and illustratesthe dosage scheme and the effect of anti-PD-L1 antibody treatment onspatial learning and memory performance using the radial arm water maze(RAWM) task at 7 months of age; black arrows indicate time points oftreatment, and illustrations indicate time points of cognitive testing;and (FIG. 17B) showing Aβ-plaque load in the cortex of anti-PD-1 treatedanimals and anti-PD-L1-treated animals compared to control animals.

FIGS. 18A-B show that PD-L1 expression increases at the CP with agingwith (FIG. 18A) showing expression of PDL1 in the CP of young (left bar)and aged (right bar) mice, measured by RT-qPCR; and (FIG. 18B) showingimmunohistochemical staining of epithelial expression of PD-L1 at the CPof young (left micrograph) and aged (right micrograph) mice. LV; lateralventricle.

FIGS. 19A-B shows thickness plots of the outer nuclear layer (ONL)throughout the entire retina measured in individual eyes of RCS ratstreated by intraperitoneal injection with anti-PD1 mAb (n=10 rats; 20eyes), IgG (n=10 rats; 20 eyes) or untreated animals (n=4 rats; 8 eyes)through histological analysis based on H&E stain with (FIG. 19A) showingall animals and (FIG. 19B) showing responders only. Data is presented asmean±standard error values; *P<0.05; **P<0.01; ***P<0.001.

FIGS. 20A-B shows thickness plots of the outer nuclear layer (ONL)throughout the entire retina measured in individual eyes of RCS ratstreated by intravitreal injection with anti-PD1 mAb (n=6) or IgG (n=5)or through histological analysis based on H&E stain with showing botheyes (the injected eye and the contralateral-non injected eye) of (FIG.20A) anti-PD1 mAb treated animals; and (FIG. 20B) IgG treated animals.Data is presented as mean±standard error values; Significant differenceswere determined through student T-test per each individual samplingpoint and are marked by asterisks (*P<0.05).

FIG. 21 shows beneficial effect of PD-1 blockade on memory performancein the DM-htau mouse model of Alzheimer's disease.

FIGS. 22A-B shows PD-1 blockade enhances hippocampal neurogenesis in5XFAD mice with (FIG. 22A) showing parasagittal brain sectionsimmunostained for neuronal marker-NeuN (in green), DCX (in red), andhoechst nuclear staining (in blue); and (FIG. 22B) showing a graphquantitating the staining in anti-PD-1 treated animals, IgG immunecontrols and aged-matched wild-type controls.

FIGS. 23A-B shows PD-1 blockade enhances hippocampal synaptic plasticityin 5XFAD mice with (FIG. 23A) showing parasagittal brain sectionsimmunostained for VgluT1 (red); and (FIG. 23B) showing a graphquantitating the staining in anti-PD-1 treated animals, IgG immunecontrols and aged-matched wild-type controls.

FIGS. 24A-B shows PD-1 blockade reduces neuronal loss in the subiculumof 5XFAD mice with (FIG. 24A) showing parasagittal brain sectionsimmunostained for neuronal marker-NeuN (in green); and (FIG. 24B)showing a graph quantitating the staining in anti-PD-1 treated animals,IgG immune controls and aged-matched wild-type controls.

DETAILED DESCRIPTION

Immune checkpoint mechanisms, which include cell-intrinsicdownregulation of activated T cell responsiveness and effector functionby inhibitory receptors, maintain systemic immune homeostasis andautoimmune tolerance (Joller et al, 2012; Pardoll, 2012). In recentyears, blockade of these immune checkpoints, such as the programmeddeath-1 (PD-1) pathway (Francisco et al, 2010), has demonstrated notableanti-tumor efficacy, highlighting the potential of unleashing the powerof the immune system in fighting various malignancies Recently, it wasshown (WO 2015/136541; Baruch et al., 2016) that administration ofanti-PD-1 antibodies to an animal model of Alzheimer's disease leads toclearance of A13, reversal of cognitive decline, and is associated withresolution of the neuroinflammatory response. Thus, systemicimmunosuppression interferes with the ability to fight off AD pathology,and by releasing restrains on the systemic immune system, AD pathologycould be mitigated.

Without wishing to be limited to any theory, immune checkpoint blockadeactivates a cascade of immunological events that starts in the peripheryand culminates in numerous activities inside the brain. Initially, animmune response increases the availability of IFN-γ at the secondarylymphoid organs (lymph nodes, spleen, etc.) and circulating monocytes inthe periphery. This immune response leads to the immunologicalactivation of the brain's choroid plexus (CP), an epithelial layer atthe brain ventricles, which forms the blood-cerebrospinal fluid-barrier(B—CSF-B), and serves as a selective gateway for leukocytes entering theCNS. The effect of the blockade of inhibitory immune checkpoints on CPgateway activity for leukocyte is mediated by the IFN-γ-inducedexpression of leukocyte trafficking molecules (adhesion molecules andchemokines) by the CP epithelium, which enables leukocyte trafficking.This increased expression leads to the recruitment of monocyte-derivedmacrophages and immunoregulatory cells to diseased sites within thebrain. Importantly, this recruitment results in a comprehensive effecton brain function, including reduced of plaque burden, restored ofimmunological balance, resolved local inflammation, reduced gliosis,reduced synaptic loss, increased neurogenesis, increased neuronalprotection and enhanced neuronal survival, collectively leading toneuroprotection and/or reduction in cognitive decline.

Immune checkpoints are molecules in the immune system that either turnup a signal (costimulatory molecules) or turn down a signal. Fourstimulatory checkpoint molecules are members of the tumor necrosisfactor (TNF) receptor superfamily—CD27, CD40, OX40, GITR and CD137.Another two stimulatory checkpoint molecules belongs to the B7-CD28superfamily—CD28 itself and ICOS. Many inhibitor checkpoint moleculesare known, including, without limitation, A2aR, B7-H3, B7-H4, BTLA,CTLA-4, IDO, KIR, LAG-3, PD-1, TIM-3 and VISTA.

The present invention provides a method for treating a disease,disorder, condition or injury of the Central Nervous System (CNS). Inone embodiment, the disclosed method for treating a disease, disorder,condition or injury of the Central Nervous System (CNS) does not includethe autoimmune neuroinflammatory disease relapsing-remitting multiplesclerosis (RRMS). The disclose method comprising administering to anindividual in need thereof an active agent that causes reduction of thelevel of systemic immunosuppression, wherein said active agent isadministered by a dosage regime comprising at least two courses oftherapy, each course of therapy comprising in sequence a treatmentsession followed by an interval session of non-treatment.

In another aspect, the present invention is directed to an active agentthat causes reduction of the level of systemic immunosuppression in anindividual, or a pharmaceutical composition comprising the active agent,for use in treating a disease, disorder, condition or injury of the CNSthat does not include the autoimmune neuroinflammatory disease,relapsing-remitting multiple sclerosis (RRMS), wherein saidpharmaceutical composition is for administration by a dosage regimencomprising at least two courses of therapy, each course of therapycomprising in sequence a treatment session followed by an intervalsession of non-treatment.

In certain embodiments, the dosage regimen is calibrated such that thelevel of systemic immunosuppression is transiently reduced.

The term “treating” as used herein refers to means of obtaining adesired physiological effect. The effect may be therapeutic in terms ofpartially or completely curing a disease and/or symptoms attributed tothe disease. The term refers to inhibiting the disease, i.e. arrestingor slowing its development; or ameliorating the disease, i.e. causingregression of the disease.

The term “systemic presence” of regulatory or effector T cells as usedherein refers to the presence of the regulatory or effector T cells (asmeasured by their level or activity) in the circulating immune system,i.e. the blood, spleen and lymph nodes. It is a well-known fact in thefield of immunology that the cell population profile in the spleen isreflected in the cell population profile in the blood (Zhao et al,2007).

The present treatment is applicable to both patients that show elevationof systemic immune suppression, as well as to patients that do not showsuch an elevation. Sometimes the individual in need for the treatmentaccording to the present invention has a certain level of peripheralimmunosuppression, which is reflected by elevated frequencies or numbersof Tregs in the circulation, and/or their enhanced functional activityand/or a decrease in IFNγ-producing leukocytes and/or decreasedproliferation of leukocytes in response to stimulation. The elevation offrequencies or numbers of Tregs can be in total numbers or as percentageof the total CD4 cells. For example, it has been found in accordancewith the present invention that an animal model of Alzheimer's diseasehas higher frequencies of Foxp3 out of CD4 cells as compared withwild-type mice. However, even if the levels of systemic Treg cells isnot elevated, their functional activity is not enhanced, the level ofIFNγ-producing leukocytes is not reduced or the proliferation ofleukocytes in response to stimulation is not decreased, in saidindividual, the method of the present invention that reduces the levelor activity of systemic immunosuppression is effective in treatingdisease, disorder, condition or injury of the CNS that does not includethe autoimmune neuroinflammatory disease RRMS. Importantly, saidsystemic immune suppression can also involve additional immune celltypes except of Tregs, such as myeloid-derived suppressor cells (MDSCs)(Gabrilovich & Nagaraj, 2009).

The level of systemic immunosuppression may be detected by variousmethods that are well known to those of ordinary skill in the art. Forexample, the level of Tregs may be measured by flow cytometry analysisof peripheral blood mononuclear cells or T lymphocytes, immunostainedeither for cellular surface markers or nuclear intracellular markers ofTreg (Chen & Oppenheim, 2011), CD45, TCR-β, or CD4 markers oflymphocytes, and measuring the amount of antibody specifically bound tothe cells. The functional activity of Tregs may be measured by variousassays; For example the thymidine incorporation assay is being commonlyused, in which suppression of anti-CD3 mAb stimulated proliferation ofCD4⁺CD25⁻ T cells (conventional T cells) is measured by [³H]thymidineincorporation or by using CFSE (5-(and 6)-carboxyfluorescein diacetatesuccinimidyl ester, which is capable of entering the cells; celldivision is measured as successive halving of the fluorescence intensityof CFSE). The number of IFNγ-producing leukocytes or their activity ortheir proliferation capacity can easily be assessed by a skilled artisanusing methods known in the art; For example, the level of IFNγ-producingleukocytes may be measured by flow cytometry analysis of peripheralblood mononuclear cells, following short ex-vivo stimulation andgolgi-stop, and immunostaining by IFNγ intracellular staining (usinge.g., BD Biosciences Cytofix/Cytoperm™ fixation/permeabilization kit),by collecting the condition media of these cells and quantifying thelevel of secreted cytokines using ELISA, or by comparing the ratio ofdifferent cytokines in the condition media, for example IL2/IL10,IL2/IL4, INFγ/TGFβ, etc. The levels of MDSCs in the human peripheralblood easily can be assessed by a skilled artisan, for example by usingflow cytometry analysis of frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+,DR⁻/LIN⁻/CD33+ and DR(−/low)/CD14+ cells, as described (Kotsakis et al,2012).

In humans, the peripheral/systemic immunosuppression may be consideredelevated when the total number of Tregs in the circulation is higherthan 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more than in ahealthy control population, the percentage of Treg cells out of thetotal CD4+ cells is elevated by 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more than in a healthy control population, or the functionalactivity of Tregs is elevated by 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more than in a healthy control population. Alternatively, theperipheral/systemic immunosuppression may be considered elevated whenthe level of IFNγ-producing leukocytes or their activity is reducedrelative to that of a healthy control population by 10, 20, 30, 40, 50,60, 70, 80, 90 or 100%; or the proliferation of leukocytes in responseto stimulation is reduced relative to that of a healthy controlpopulation by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

An agent may be considered an agent that causes reduction of the levelof systemic immunosuppression when, upon administration of the agent toan individual, the total number of Tregs in the circulation of thisindividual is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% ascompared with the level before administration of the agent, thepercentage of Treg cells out of the total CD4+ cells drops by 10, 20,30, 40, 50, 60, 70, 80, 90 or 100% relative to that of a healthy controlpopulation or the functional activity of Tregs is reduced by 10, 20, 30,40, 50, 60, 70, 80, 90 or 100% as compared with the level beforeadministration of the agent. Alternatively, an agent may be consideredan agent that causes reduction of the level of systemicimmunosuppression when, upon administration of the agent to anindividual, the total number of IFNγ-producing leukocytes or theiractivity is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% ormore; or the proliferation of leukocytes in response to stimulation isincreased relative to that of a healthy control population by 10, 20,30, 40, 50, 60, 70, 80, 90 or 100% or more.

In certain embodiments, the active agent causes reduction of the levelof systemic immunosuppression by release of a restraint imposed on theimmune system by one or more immune checkpoints, for example by blockadeof the one or more immune checkpoints.

In certain embodiments, the reduction of the level of systemicimmunosuppression is associated with an increase in systemic presence oractivity of IFNγ-producing leukocytes.

In certain embodiments, the active agent causes reduction of the levelof systemic immunosuppression and thereby an increase in the systemicpresence or activity of effector T cells.

In certain embodiments, the reduction of the level of systemicimmunosuppression is associated with an increase in systemic presence oractivity of an IFNγ cytokine.

In certain embodiments, the reduction of the level of systemicimmunosuppression is associated with a decrease in systemic presence oractivity of regulatory T-cells.

In certain embodiments, the reduction of the level of systemicimmunosuppression is associated with a decrease in systemic presence oractivity of an IL-10 cytokine.

In certain embodiments, the reduction of the level of systemicimmunosuppression is associated with a decrease in systemic presence oractivity of myeloid-derived suppressor cells (MDSCs).

In certain embodiments, the active agent causes reduction of the levelof systemic immunosuppression and thereby an increase in the systemicpresence or activity of effector T cells.

The checkpoints that may be manipulated to release the systemicimmunosuppression are referred to herein as a pair of an immunecheckpoint receptor and its native ligand or either one of the twopartners. For example, PD-1, which has two known ligands is referred toherein as “PD-L1” and “PD-L2”, while B7H3, the ligand of which has notyet been identified, is referred to simply by “B7H3”. The checkpointsthat may be manipulated to release the systemic immunosuppression inaccordance with the present invention include, without limitation,PD-1-PD-L1, PD-1-PD-L2, CD28-CD80, CD28-CD86, CTLA-4-CD80, CTLA-4-CD86,ICOS-B7RP1, B7H3, B7H4, B7H7, B7-CD28-like molecule, BTLA-HVEM, KIR-MHCclass I or II, LAG3-MHC class I or II, CD137-CD137L, OX40-OX40L,CD27-CD70, CD40L-CD40, TIM3-GAL9, V-domain Ig suppressor of T cellactivation (VISTA), STimulator of INterferon Genes (STING), T cellimmunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain(TIGIT), glucocorticoid-induced tumor necrosis factor receptor relatedprotein (GITR), A2aR-Adenosine and indoleamine-2,3-dioxygenase(IDO)-L-tryptophan.

Agents capable of blocking immune checkpoints are known in the art(Colombo & Piconese, 2007) and these agents can be used in accordancewith the present invention. Each one of the cited publications below,and Pardoll, 2012, is incorporated by reference as if fully disclosedherein.

In certain embodiments, the active agent that may be used according tothe present invention may be an antibody. An antibody as disclosedherein can be a polyclonal antibody, a monoclonal antibody, a dimer, amultimer, a multispecific antibody, a human antibody, a humanizedantibody, a recombinant antibody, a chimeric antibody, bi-functionalantibody, a cell-associated antibody like an Ig receptor, a linearantibody, a diabody, a minibody or a nanobody, so long as the fragmentexhibits the desired biological activity, and single chain derivativesof the same. An antibody can be a full-length immunoglobulin moleculecomprising the VH and VL domains, as well as a light chain constantdomain (CL) and heavy chain constant domains, CH1, CH2 and CH3, or animmunologically active fragment of a full-length immunoglobulinmolecule, such as, e.g., a single domain antibody (sdAb), a single-chainvariable fragment (scFv), a Fab fragment, a F(ab′)2 fragment, a Fcfragment, a Fd fragment, a Fv fragment. An antibody can be derived fromany vertebrate species (e.g., human, goat, horse, donkey, murine, rat,rabbit, or chicken), and can be of any type (e.g., IgG, IgE, IgM, IgD,and IgA), class (e.g., IgA, IgD, IgE, IgG, and IgM) or subclass (IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2). Functionally, an antibody disclosedherein may be an antagonist antibody, meaning an antibody that inhibitsa biological activity or an antibody disclosed herein may be an agonistantibody, meaning an antibody that stimulates a biological activity.Similarly, an antibody disclosed herein may be a neutralizing antibody,meaning an antibody that can block or neutralize a biological activity.For general disclosure on the structure of naturally occurringantibodies, non-naturally occurring antibodies, and antigeniccompound-binding fragments thereof, see, e.g., Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994); Borrabeck, AntibodyEngineering, 2d ed. (Oxford University Press 1995), each of which ishereby incorporated by reference in its entirety.

An antibody disclosed herein may be, without limitation, an anti-PD-1,an anti-PD-L1, an anti-PD-L2, an anti-CTLA-4, an anti-CD80, ananti-CD86, an anti-B7RP1, an anti-B7-H3, an anti-B7-H4, an anti-B7-H7,an anti-BTLA, an anti-HVEM, an anti-CD-27, an anti-CD40, an anti-CD40L,an anti-CD70, an anti-CD80, an anti-CD86, an anti-CD137, an anti-CD137L,an anti-OX40, an anti-OX40L, an anti-TIM-3, an anti-Galectin9, ananti-KIR, an anti-LAG-3, an anti-ICOS, an anti-VISTA, an anti-STING, ananti-TIGIT, anti-GITR or any combination thereof. An antibody disclosedherein may be administered to a human at a dosage of for example about0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5mg/kg, 0.2 mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2mg/kg-6 mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15mg/kg, 0.3 mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg,1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5mg/kg.

Programmed cell death protein 1, also known as PD-1 and CD279 (clusterof differentiation 279), is a cell surface receptor that belongs to theimmunoglobulin superfamily and is expressed on T cells and pro-B cells.PD-1 binds two ligands, PD-L1 and PD-L2. Functioning as an immunecheckpoint, PD-1 plays an important role in down regulating the immunesystem by preventing the activation of T-cells, which in turn reducesautoimmunity and promotes self-tolerance. The inhibitory effect of PD-1is accomplished through a dual mechanism of promoting apoptosis(programmed cell death) in antigen specific T-cells in lymph nodes whilesimultaneously reducing apoptosis in regulatory T cells (suppressor Tcells). As such, compounds which inhibit PD-1 function, such as PD-1inhibitors, PD-L1 inhibitors and/or PD-L2 inhibitors, serve to activatethe immune system. One class of PD-1 inhibitors includes antagonist orneutralizing anti-PD-1, anti-PD-L1 and anti-PD-L2 antibodies. Manyantagonist or neutralizing anti-PD-1, anti-PD-L1 and anti-PD-L2antibodies are known in the art. For example, the anti-PD-1 antibodyused in accordance with the present invention may be selected from thosedisclosed in Ohaegbulam et al. (Ohaegbulam et al, 2015), the entirecontents of which being hereby incorporated herein by reference.Examples of human or humanized anti-PD-1 antibodies include, withoutlimitation, CD279 (human anti-PD1 monoclonal antibody, Bio X Cell),MEDI0680 (AMP-514; humanized IgG4 anti-PD-1 monoclonal antibody;AstraZeneca), Nivolumab (BMS-936558; human IgG4 anti-PD1 monoclonalantibody; Bristol-Myers Squibb), Pembrolizumab (Lambrolizumab, MK-3475;humanized IgG4 anti-PD1 monoclonal antibody; Merck), Pidilizumab(CT-011; humanized IgG1 anti-PD1 monoclonal antibody; Medivation) andTSR-042 (humanized IgG4 anti-PD-1 monoclonal antibody; Tesaro). Examplesof human or humanized anti-PD-L1 antibodies include, without limitation,Avelumab (MSB0010718C; human IgG1 anti-PD-L1 monoclonal antibody;Merck-Serono), Atezolizumab (MPDL3280A, RG7446; human IgG anti-PD-L1monoclonal antibody; Hoffmann-La Roche), BMS-936559 (MDX-1105; humanIgG4 anti-PD-L1 monoclonal antibody; Bristol-Myers Squibb), Durvalumab(MED14736; humanized IgG1 anti-PD-L1 monoclonal antibody; AstraZeneca),KN035 (anti-PD-L1 monoclonal antibody; 3D Medicines) and LY3300054(anti-PD-L1 monoclonal antibody; Eli Lilly). Examples of human orhumanized anti-PD-L2 antibodies include, without limitation, AMP-224(IgG2a Fc fusion protein of PD-L2; AstraZeneca). In certain embodiments,an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-PD-L2antibody may be administered to a human at a dosage of for example about0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5mg/kg, 0.2 mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2mg/kg-6 mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15mg/kg, 0.3 mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg,1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5mg/kg.

In certain embodiments, Pidilizumab may be administered to a human at adosage of 0.2-6 mg/kg or between 1.5-6 mg/kg; Pembrolizumab may beadministered to a human at a dosage of 1-10 mg/kg; Nivolumab may beadministered to a human at a dosage of 0.3-20 mg/kg, 0.3-10 mg/kg, 1-10mg/kg or at 1 or 3 mg/kg; BMS-936559 may be administered to a human at adosage of 0.3-10 mg/kg; Atezolizumab may be administered to a human at adosage of 1-20 mg/kg; Durvalumab may be administered to a human at adosage of 0.1-15 mg/kg; and Avelumab may be administered to a human at adosage of 1-20 mg/kg.

T-cell immunoglobulin and mucin domain-3 (TIM-3) is a Th1-specific cellsurface protein that acts as an immune checkpoint that inhibitslymphocyte activity by down regulating macrophage activation and playingan important role in CD8+ T cell exhaustion that takes place in chronicimmune conditions. TIM-3 acts as a negative regulator of Th1/Tc1function by triggering cell death upon interaction with its ligand,galectin-9 (Gal9). As such, compounds which inhibit TIM-3 function, suchas TIM-3 inhibitors and/or Gal9 inhibitors, serve to activate the immunesystem. One class of TIM-3 inhibitors includes antagonist orneutralizing antibodies against TIM-3 and/or Gal-9. Many antagonist orneutralizing anti-TIM-3 and anti-Gal9 antibodies are known in the art.Examples of human or humanized anti-TIM-3 antibodies include, withoutlimitation, AF2365 (human IgG anti-TIM-3 monoclonal antibody; R&DSystems), CD366 (human IgG1 anti-TIM-3 monoclonal antibody; BioLegend),F38-2E2 (human IgG1 anti-TIM-3 monoclonal antibody; R&D Systems), L3D(human IgG1 anti-TIM-3 monoclonal antibody; CN 102492038 B), MAB2365(human IgG2a anti-TIM-3 monoclonal antibody; R&D Systems), MAB23651(human IgG1 anti-TIM-3 monoclonal antibody; R&D Systems) and TSR-022(humanized IgG4 anti-TIM-3 monoclonal antibody; Tesaro). In certainembodiments, an anti-TIM-3 antibody and/or an anti-Gal9 antibody may beadministered to a human at a dosage of for example about 0.1 mg/kg-20mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5 mg/kg, 0.2mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2 mg/kg-6mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15 mg/kg, 0.3mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg,1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5 mg/kg.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known ascluster of differentiation 152 (CD152), is a protein receptorfunctioning as an immune checkpoint that downregulates immune responses.CTLA-4 is constitutively expressed in Tregs but only upregulated inconventional T cells after activation. CTLA-4 acts as an “off” switchwhen bound to CD80 or CD86 on the surface of antigen-presenting cells.As such, compounds which inhibit CTLA-4 function, such as CTLA-4inhibitors, CD80 inhibitors and/or CD86 inhibitors, serve to activatethe immune system. One class of CTLA-4 inhibitors includes antagonist orneutralizing antibodies against CTLA-4, CD80 and/or CD86. Manyantagonist or neutralizing anti-CTLA-4, anti-CD80 and anti-CD86antibodies are known in the art. Examples of human or humanizedanti-CTLA-4 antibodies include, without limitation, Ipilimumab (humanIgG1 anti-CTLA-4 monoclonal antibody; Bristol-Myers Squibb) andTremelimumab (human IgG2 anti-CTLA-4 monoclonal antibody; Pfizer). Incertain embodiments, an anti-CTLA-4 antibody, an anti-CB80 antibodyand/or an anti-CD86 antibody may be administered to a human at a dosageof for example about 0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15 mg/kg, 0.1mg/kg-10 mg/kg, 0.1 mg/kg-5 mg/kg, 0.2 mg/kg-20 mg/kg, 0.2 mg/kg-15mg/kg, 0.2 mg/kg-10 mg/kg, 0.2 mg/kg-6 mg/kg, 0.2 mg/kg-5 mg/kg, 0.3mg/kg-20 mg/kg, 0.3 mg/kg-15 mg/kg, 0.3 mg/kg-10 mg/kg, 0.3 mg/kg-5mg/kg, 1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1 mg/kg-5mg/kg, 1.5 mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10 mg/kg, 1.5mg/kg-6 mg/kg or 1.5 mg/kg-5 mg/kg.

Killer-cell immunoglobulin-like receptors (KIRs) are a family of type Itransmembrane glycoproteins expressed on the plasma membrane of naturalkiller (NK) cells and a minority of T cells. They regulate the killingfunction of these cells by interacting with major histocompatibility(MHC) class I molecules, which are expressed on all nucleated celltypes. Thus, KIRs are inhibitors of lymphocyte activity. As such,compounds which inhibit KIR function, such as KIR inhibitors, serve toactivate the immune system. One class of KIR inhibitors includesantagonist or neutralizing antibodies against KIR. Many antagonist orneutralizing anti-KIR antibodies are known in the art. Examples of humanor humanized anti-KIR antibodies include, without limitation, Lirilumab(BMS-986015; human anti-KIR monoclonal antibody; Bristol-Myers Squibb).In certain embodiments, an anti-KIR antibody may be administered to ahuman at a dosage of for example about 0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5 mg/kg, 0.2 mg/kg-20 mg/kg, 0.2mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2 mg/kg-6 mg/kg, 0.2 mg/kg-5mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15 mg/kg, 0.3 mg/kg-10 mg/kg, 0.3mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5 mg/kg.

Lymphocyte-activation gene 3 (LAG-3), also known as cluster ofdifferentiation 223 (CD223), is a cell surface molecule with diversebiologic effects on T cell function. LAG-3 is an immune checkpoint thatinhibits lymphocyte activity by suppressing an immune response by actionto Tregs as well as direct effects on CD8+ T cells. As such, compoundswhich inhibit LAG-3 function, such as LAG-3 inhibitors, serve toactivate the immune system. One class of LAG-3 inhibitors includesantagonist or neutralizing antibodies against LAG-3. Many antagonist orneutralizing anti-LAG-3 antibodies are known in the art. Examples ofhuman or humanized anti-LAG-3 antibodies include, without limitation,BMS-986016 (human anti-LAG-3 monoclonal antibody; Bristol-Myers Squibb).In certain embodiments, an anti-LAG-3 antibody may be administered to ahuman at a dosage of for example about 0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5 mg/kg, 0.2 mg/kg-20 mg/kg, 0.2mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2 mg/kg-6 mg/kg, 0.2 mg/kg-5mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15 mg/kg, 0.3 mg/kg-10 mg/kg, 0.3mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5 mg/kg.

OX40, also known as cluster of differentiation 134 (CD134), is a memberof the TNFR-superfamily of receptors. OX40 promotes the expansion ofeffector and memory T cells, however it is also noted for its ability tosuppress the differentiation and activity of T-regulatory cells, andalso for its regulation of cytokine production. Being transientlyexpressed after T-cell receptor engagement, OX40 is only upregulated onthe most recently antigen-activated T cells within inflammatory lesions.Its ligand is OX40L, also known as cluster of differentiation 252(CD252). As such, compounds which activate or stimulate OX40 function,such as OX40 activators and/or OX40L activators, serve to activate theimmune system. One class of OX40 activators includes agonist antibodiesagainst OX40 and OX40L. Many agonist antibodies against OX40 and/orOX40L are known in the art. Examples of human or humanized anti-OX40antibodies include, without limitation, GSK3174998; humanized IgG1anti-OX40 monoclonal antibody; GlaxoSmithKline), MED10562 (humanizedanti-OX40 monoclonal antibody; MedImmune) and MED16383 (human OX40fusion protein; MedImmune). Other anti-OX40 antibodies include, withoutlimitation, MED16469 (9B12; murine anti-OX40 monoclonal antibody;MedImmune). In certain embodiments, an anti-OX40 antibody and/or ananti-OX40L antibody may be administered to a human at a dosage of forexample about 0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10mg/kg, 0.1 mg/kg-5 mg/kg, 0.2 mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2mg/kg-10 mg/kg, 0.2 mg/kg-6 mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20mg/kg, 0.3 mg/kg-15 mg/kg, 0.3 mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6mg/kg or 1.5 mg/kg-5 mg/kg.

Anti-GITR antibodies target glucocorticoid-induced tumor necrosis factorreceptor related protein (GITR), which is regularly expressed on thesurface of regulatory T-cells (Tregs) and is expressed on the surface ofeffector T-cells after their activation. Anti-GITR antibodies block theinteraction of GITR, found on multiple types of T cells, with itsligand, thereby inducing both the activation of tumor-antigen-specific Teffector cells, as well as abrogating the suppression induced byinappropriately activated T regulatory cells. As such, compounds whichactivate or stimulate GITR function, such as GITR activators, serve toactivate the immune system. One class of GITR activators includesagonist antibodies against GITR. Many agonist antibodies against GITRare known in the art. Examples of human or humanized anti-TIGRantibodies include, without limitation, GWN323 (humanized anti-GITRmonoclonal antibody; Novartis) and TRX518 (humanized anti-GITRmonoclonal antibody; GITR, Inc.). In certain embodiments, an anti-GITRantibody may be administered to a human at a dosage of for example about0.1 mg/kg-20 mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5mg/kg, 0.2 mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2mg/kg-6 mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15mg/kg, 0.3 mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1mg/kg-15 mg/kg, 1 mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg,1.5 mg/kg-15 mg/kg, 1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5mg/kg.

CD27 is a member of the tumor necrosis factor receptor superfamily. CD27activity is governed by the transient availability of its ligand, CD70,on lymphocytes and dendritic cells. Activation of CD27 plays a key rolein regulating B-cell activation and immunoglobulin synthesis, supportsantigen-specific expansion of naïve T cells, is required for generationand long-term maintenance of T cell immunity and is a memory marker of Bcells. CD27 transduces signals that lead to the activation of NF-κB andMAPK8/JNK. As such, compounds which activate or stimulate CD27 function,such as CD27 activators and/or CD70 activators, serve to activate theimmune system. One class of CD27 activators includes agonist antibodiesagainst CD27 and/or CD70. Many agonist antibodies against CD27 and/orCD70 are known in the art. Examples of human or humanized anti-CD27antibodies include, without limitation, Varlilumab (CDX-1127; humananti-CD27 monoclonal antibody; Celldex Therapeutics). In certainembodiments, an anti-CD27 antibody and/or an anti-CD70 antibody may beadministered to a human at a dosage of for example about 0.1 mg/kg-20mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5 mg/kg, 0.2mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2 mg/kg-6mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15 mg/kg, 0.3mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg,1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5 mg/kg.

Inducible T-cell COStimulator (ICOS), also known as cluster ofdifferentiation 278 (CD278), is a CD28-superfamily costimulatorycell-surface receptor that is expressed on activated T cells. It is anactivator of T cell function. As such, compounds which activate orstimulate ICOS function, such as ICOS activators or B7RP1 activators,serve to activate the immune system. One class of ICOS activatorsincludes agonist antibodies against ICOS and/or B7RP1. Many agonistanti-ICOS and anti-B7RP1 antibodies are known in the art. In certainembodiments, an anti-ICOS antibody and/or an anti-B7RP1 antibody may beadministered to a human at a dosage of for example about 0.1 mg/kg-20mg/kg, 0.1 mg/kg-15 mg/kg, 0.1 mg/kg-10 mg/kg, 0.1 mg/kg-5 mg/kg, 0.2mg/kg-20 mg/kg, 0.2 mg/kg-15 mg/kg, 0.2 mg/kg-10 mg/kg, 0.2 mg/kg-6mg/kg, 0.2 mg/kg-5 mg/kg, 0.3 mg/kg-20 mg/kg, 0.3 mg/kg-15 mg/kg, 0.3mg/kg-10 mg/kg, 0.3 mg/kg-5 mg/kg, 1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, 1mg/kg-10 mg/kg, 1 mg/kg-5 mg/kg, 1.5 mg/kg-20 mg/kg, 1.5 mg/kg-15 mg/kg,1.5 mg/kg-10 mg/kg, 1.5 mg/kg-6 mg/kg or 1.5 mg/kg-5 mg/kg.

In certain embodiments, combinations of antibodies may be used such asbut not limited to: CT-011 in combination with Rituximab (trade namesRituxan, MabThera and Zytux) a chimeric monoclonal antibody against theprotein CD20, for example, each at 3 mg/kg; Nivolumab (for example 1mg/kg) in combination with Ipilimumab; for example at 3 mg/kg); orNivolumab (e.g. 1-10 mg/kg) in combination with a anHLA-A*0201-restricted multipeptide vaccine (Weber et al, 2013).

In certain embodiments, the active agent that may be used according tothe present invention may be an antibody mimetic. Antibody mimetics canspecifically bind antigens like antibodies, but that are notstructurally related to antibodies. They are usually artificial peptidesor proteins with a molar mass of about 3 kDa to 20 kDa. In aspects ofthis embodiment, an antibody mimetic disclosed herein may be an affibodymolecule; an affilin; an affimer; an affitin; an alphabody; ananticalin; an avimer; a DARPin; a fynomer; a Kunitz domain peptide; or amonobody. Non-limiting examples of antibody mimetics are presented inTable 1.

TABLE 1 Antibody mimetics Antibody Example mimetic Scaffold Molar massdrug Affibodies^([1]) Z domain of Protein A 6 kDa ABY-025 Affilins^([2])Gamma-B crystallin 20 Da Ubiquitin 10 Da SPVF 2801 Affimers^([3])Cystatin 12-14 kDa Affitins^([4]) Sac7d (from Sulfolobus 7 kDaacidocaldarius) Alphabodies^([5]) Triple helix coiled coil 10 DaCMPX-1023 Anticalins^([6]) Lipocalins 20 Da Avimers^([7]) A domains ofvarious 9-18 kDa membrane receptors DARPins^([8]) Ankyrin repeat motif10-19 kDa MP0112 Fynomers^([9]) SH3 domain of Fyn 7 kDa Kunitz domainKunitz domains of various 6 kDa Ecallantide peptides^([10]) proteaseinhibitors (Kalbitor) Monobodies^([11]) 10^(th) type III 10 kDaPeqdinetanib domain of fibronectin (Anqiocept) ^([1])Nygren PA (June2008); ^([2])Ebersbach et al. (2007); ^([3])Johnson et al. (2012);^([4])Krehenbrink et al.(2008); ^([5])Desmet et al. (2014); ^([6])SkerraA (2008); ^([7])Silverman et al. (2005); ^([8])Stumpp et al. (2008);^([9])Grabulovski et al. (2007); ^([10])Nixon et al. (2006);^([11])Koide et al. (2007).

In certain embodiments, the active agent that may be used according tothe present invention may be an aptamer. Aptamers are oligonucleotide orpeptide molecules that bind to a specific target molecule. In aspects ofthis embodiment, an aptamer disclosed herein may be a DNA aptamer, a RNAaptamer, a XNA aptamer or a peptide aptamer.

In certain embodiments, an antibody disclosed herein may be incombination with an adjuvant. An adjuvant is any substance or mixture ofsubstances that increases or diversifies the immune response. Anadjuvant may serve to reduce the number of immunizations or the amountof antigen required for protective immunization. Non-limiting adjuvantsinclude, e.g., liposomes, oily phases, including, without limitation,the Freund type of adjuvants, such as, e.g., Freund's complete adjuvant(FCA); Freund's incomplete adjuvant (FIA); sapogenin glycosides, suchas, e.g., saponins; carbopol; N-acetylmuramyl-L-alanyl-D-isoglutamine(commonly known as muramyl dipeptide or “MDP”); and lipopolysaccharide(LPS). Such adjuvants are generally used in the form of an emulsion withan aqueous phase, or, more commonly, with water-insoluble inorganicsalts. These inorganic salts include aluminum hydroxide, zinc sulfate,colloidal iron hydroxide, calcium phosphate or calcium chloride. Inaspects of this embodiment, an antibody disclosed herein may be combinedwith, e.g., an anti-CTLA-4 antibody in combination with an anti-OX40antibody and a TLR9 ligand such as CpG (Marabelle et al, 2013).

In certain embodiments, the active agent that may be used according tothe present invention may be a small molecule. In aspects of thisembodiment, a small molecule disclosed herein may be: (a) a p300inhibitor (Liu et al, 2013), such as gemcitabine (low dose) or C646 oranalogs thereof, i.e. a compound of the formula I:

wherein R₁ is selected from H, —CO₂R₆, —CONR₆R₇, —SO₃H, or —SO₂NR₆R₇; R₂is selected from H, —CO₂R₆, or halogen, preferably Cl; R₃ is selectedfrom halogen, preferably F, —NO₂, —CN, —CO₂R₆, preferably CO₂CH₃ orCO₂CH₂CH₃, or —CH₂OH; R₄ and R₅ each independently is H or —C₁-C₆ alkyl,preferably methyl; R₆ is H or —C₁-C₆ alkyl, preferably H, methyl orethyl; and R₇ is H or —C₁-C₆ alkyl, preferably H or methyl; (b) aSunitinib; (c) a Polyoxometalate-1 (POM-1) (Ghiringhelli et al, 2012);(d) an α,β-methyleneadenosine 5′-diphosphate (APCP); (e) an arsenictrioxide (As₂O₃); (f) a GX15-070 (Obatoclax); (g) a retinoic acidantagonist such as Ro 41-5253 (a synthetic retinoid and selective smallmolecule antagonist) or LE-135; (h) an SIRPα (CD47) antagonist, such asCV1-hIgG4 (SIRPα variant) as sole agent or in combination with anti-CD47antibody; (i) a CCR4 antagonist, such as AF399/420/18025 as sole agentor in combination with anti-CCR4 antibody; (j) an adenosine receptorantagonist; (k) an adenosine A1 receptor antagonist; an adenosine A2areceptor; (m) an adenosine A2b receptor antagonist; (n) an A3 receptorantagonist; (o) an antagonist of indoleamine-2,3-dioxygenase; or (p) anHIF-1 regulator.

In certain embodiments, the agent is a p300 inhibitor, which formulasare listed in Table 2, i.e. C646(4-(4-((5(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoicacid), C146(4-hydroxy-3-(((2-(3-iodophenyl)benzo[d]oxazol-5-yl)imino)methyl)benzoicacid) or C375(2-chloro-4-(5-((2,4-dioxo-3-(2-oxo-2-(p-tolylamino)ethyl)thiazolidin-5-ylidene)methyl)furan-2-yl)benzoicacid). In particular, the p300 inhibitor is C646.

TABLE 2*

C646

C375

C146 *Based on Bowers et al. (2010)

In certain embodiments, the adenosine receptor antagonist may beCGS15943(9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine); theadenosine A1 receptor antagonist may be PSB 36(1-Butyl-8-(hexahydro-2,5-methanopentalen-3a(1H)-yl)-3,7-dihydro-3-(3-ydroxypropyl)-1H-purine-2,6-dione);the adenosine A2a receptor antagonist may be SCH58261(5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine),SYN115(4-Hydroxy-N-[4-methoxy-7-(4-morpholinyl)-2-benzothiazolyl]-4-methyl-1-piperidinecarboxamide),FSPTP (also called SCH58261(5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ε]-1,2,4-triazolo[1,5-c]pyrimidine),SCH442416(2-(2-Furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine),or ZM241385 (also called tozadenant(4-Hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide);the adenosine A2b receptor antagonist may be PSB 603(8-{4-[4-(4-chlorophenyl)piperazine-1-sulfonyl]phenyl}-1-propyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione(Nakatsukasa et al, 2011)); and the A3 receptor antagonist may beMRS3777 (2-Phenoxy-6-(cyclohexylamino)purine hemioxalate).

In certain embodiments, the small molecule inhibitor of theindoleamine-2,3-dioxygenase pathway may be Indoximod(NSC-721782/NLG-9189 (1-Methyl-D-tryptophan), NewLink Genetics),INCB024360((4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine,Incyte) or NLG-919(1-Cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol), NewLinkGenetics).

The HIF-1 regulator may be M30,(5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline) described inZheng et al. (Zheng et al, 2015).

In certain embodiments, an active agent that may be used according tothe present invention may be any combination of an antibody disclosedherein and a small molecule disclosed herein. In aspects of thisembodiment, an active agent may be any combination of antibody disclosedherein and small molecule disclosed herein.

In certain embodiments, an active agent that may be used according tothe present invention may be a protein selected from the groupconsisting of: (a) Neem leaf glycoprotein (NLGP; (Roy et al, 2013));and/or (b) sCTLA-4 (soluble isoform of CTLA-4) (Ward et al, 2013).

In certain embodiments, an active agent that may be used according tothe present invention may be a silencing molecule. In aspects of thisembodiment, a silencing molecule is selected from the group consistingof miR-126 antisense (Qin et al, 2013) and anti-galectin-1 (Gal-1;(Dalotto-Moreno et al, 2013)).

In certain embodiments, an active agent that may be used according tothe present invention may be an OK-432 (lyophilized preparation ofStreptococcus pyogenes) (Hirayama et al, 2013).

In certain embodiments, an active agent that may be used according tothe present invention may be a combination of IL-12 and anti-CTLA-4.

In certain embodiments, the agent can be derived from a broad spectrumof antibiotics which targets gram-positive and gram-negative bacteria,and thereby facilitating immunomodulation of Tregs, e.g. vancomycinwhich targets gram-positive bacteria and has been shown to reduce Treglevels/activity (Brestoff & Artis, 2013; Smith et al, 2013).

In certain embodiments, an active agent that may be used according tothe present invention may be any combination of an antibody disclosedherein, an antibody mimetic disclosed herein, an aptamer disclosedherein, a small molecule disclosed herein, a Neem leaf glycoproteindisclosed herein, a sCTLA-4 disclosed herein, a silencing moleculedisclosed herein, an OK-432 disclosed herein, and/or a combination ofIL-12 and anti-CTLA-4 disclosed herein.

As stated above, the active agent is administered by a dosage regimecomprising at least two courses of therapy, each course of therapycomprising in sequence a treatment session followed by an intervalsession of non-treatment.

The term “treatment session” is used interchangeably herein with theterms “treatment period” or “period of treatment” and refers to asession during which one or more active agents disclosed herein areadministered to the individual being treated. As discussed in moredetail below, a treatment session can be a single dosing event, or canbe a multiple dosing regimen that occurs over a period of time. Atreatment session results in a therapeutically effective amount of anactive agent disclosed herein to be consistently maintained throughoutthe treatment session.

The term “non-treatment session” is used interchangeably herein with theterms “non-treatment period”, “period of no treatment”, “intervalsession” or “interval session of non-treatment” and refers to a periodof time during which no active agent disclosed herein is administered tothe individual being treated. The cessation of active agentadministration during the non-treatment session results in the reductionof an active agent disclosed herein to sub-therapeutic levels in theindividual being treated. As disclosed herein, a “non-treatment session”is not the same event as a period of time that intervenes between adosing event making up a multiple dosing regimen that occurs over aperiod of time during a treatment session. If administration of one ormore active agents disclosed herein during a treatment session is arepeated administration, the non-treatment session is longer than theintervening period between these repeated administrations during thetreatment session.

The dosage regime may be determined in a number of ways. For example,the level of immunosuppression may be calibrated to a desired level foreach patient who is being treated (personalized medicine), by monitoringthe level or activity of IFN-γ-producing leukocytes or proliferationrate of leukocytes in response to stimulation individually, andadjusting the treatment session, the frequency of administration and theinterval session empirically and personally as determined from theresults of the monitoring.

Thus, the treatment session may comprise administering the active agentor pharmaceutical composition to the individual and the treatmentsession is maintained at least until the systemic presence or level ofIFN-γ-producing leukocytes, or the rate of proliferation of leukocytesin response to stimulation rises above a reference, the administering ispaused during the interval session, and the interval session ismaintained as long as the level is above the reference, wherein thereference is selected from (a) the level of systemic presence oractivity of IFN-γ-producing leukocytes, or the rate of proliferation ofleukocytes in response to stimulation, measured in the most recent bloodsample obtained from said individual before said administering; or (b)the level of systemic presence or activity of IFN-γ-producingleukocytes, or the rate of proliferation of leukocytes in response tostimulation, characteristic of a population of individuals afflictedwith a disease, disorder, condition or injury of the CNS.

The length of the treatment and non-treatment or interval sessions maybe determined by physicians in clinical trials directed to a certainpatient population and then applied consistently to this patientpopulation, without the need for monitoring the level ofimmunosuppression on a personal basis.

In certain embodiments, the treatment session comprises administeringthe active agent to the individual and the treatment session ismaintained at least until the systemic presence of the active agentreaches therapeutic levels, the administering is paused during theinterval session, and the interval session is maintained as long as thelevel is above about 95%, 90%, 80%, 70%, 60% or 50% of said therapeuticlevel. The term “therapeutic level” as used herein refers to generallyaccepted systemic levels of drugs used to block immune checkpoints inknown therapies, such as cancer therapy (see above).

In certain embodiments, the treatment session comprises administeringthe active agent to the individual and the treatment session ismaintained at least until the systemic presence or activity of theactive agent reaches a therapeutic level, at which point theadministration is then stopped, and a non-treatment period is maintainedas long as the systemic presence or activity of the active agent ismaintained above a threshold therapeutic level. In aspects of thisembodiment, a threshold therapeutic level is a level that is, e.g. atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, of the therapeuticlevel. The term “therapeutic level” as used herein refers to generallyaccepted systemic levels of drugs used to block immune checkpoints inknown therapies, such as cancer therapy (see above). In aspects of thisembodiment, the active agent is an anti-PD-1 antibody, an anti-PD-L1antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-B7RP1antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-B7-H7antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-CD-27antibody, an anti-CD40 antibody, an anti-CD40L antibody, an anti-CD70antibody, an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD137antibody, an anti-CD137L antibody, an anti-OX40 antibody, an anti-OX40Lantibody, an anti-TIM-3 antibody, an anti-Galectin9 antibody, ananti-KIR antibody, an anti-LAG-3 antibody, an anti-ICOS antibody, ananti-VISTA antibody, an anti-STING, an anti-TIGIT, anti-GITR or anycombination thereof.

In certain embodiments, the treatment session comprises administeringthe active agent to the individual and the treatment session ismaintained at least until the systemic presence or activity of theactive agent reaches a therapeutic level, at which point theadministration is then stopped, and a non-treatment period is maintainedas long as a beneficial effect on cognition is maintained above thelevel before treatment commencement. In aspects of this embodiment, abeneficial effect on cognition is maintained is one that shows animprovement of, e.g. at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, above the cognition level before treatment commencement. In aspectsof this embodiment, the active agent is an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, ananti-B7RP1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, ananti-B7-H7 antibody, an anti-BTLA antibody, an anti-HVEM antibody, ananti-CD-27 antibody, an anti-CD40 antibody, an anti-CD40L antibody, ananti-CD70 antibody, an anti-CD80 antibody, an anti-CD86 antibody, ananti-CD137 antibody, an anti-CD137L antibody, an anti-OX40 antibody, ananti-OX40L antibody, an anti-TIM-3 antibody, an anti-Galectin9 antibody,an anti-KIR antibody, an anti-LAG-3 antibody, an anti-ICOS antibody, ananti-VISTA antibody, an anti-STING, an anti-TIGIT, anti-GITR or anycombination thereof.

In certain embodiments, the treatment session comprises administeringthe active agent to the individual and the treatment session ismaintained at least until the systemic presence or activity of theactive agent reaches a therapeutic level, at which point theadministration is then stopped, and a non-treatment period is maintainedas long as a beneficial effect on vision is maintained above the levelbefore treatment commencement. In aspects of this embodiment, abeneficial effect on vision is maintained is one that shows animprovement of, e.g. at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, above the vision level before treatment commencement. In aspects ofthis embodiment, the active agent is an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, ananti-B7RP1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, ananti-B7-H7 antibody, an anti-BTLA antibody, an anti-HVEM antibody, ananti-CD-27 antibody, an anti-CD40 antibody, an anti-CD40L antibody, ananti-CD70 antibody, an anti-CD80 antibody, an anti-CD86 antibody, ananti-CD137 antibody, an anti-CD137L antibody, an anti-OX40 antibody, ananti-OX40L antibody, an anti-TIM-3 antibody, an anti-Galectin9 antibody,an anti-KIR antibody, an anti-LAG-3 antibody, an anti-ICOS antibody, ananti-VISTA antibody, an anti-STING, an anti-TIGIT, anti-GITR or anycombination thereof.

In certain embodiments, the treatment session may be a singleadministration or it may comprise multiple administrations given duringa prescribed period of time. In aspects of this embodiment, a treatmentsession may be multiple administrations given in the course of between,e.g., 1 day to four weeks, 2 days to four weeks, 3 days to four weeks, 4days to four weeks, 5 days to four weeks, 6 days to four weeks, one weekand four weeks, 10 days and four weeks, two weeks and four weeks, 17days and four weeks or three weeks and four weeks. For example, thetreatment session may comprise two administrations both given within oneweek, such as, e.g., the second administration given 1, 2, 3, 4, 5 or 6days after the first administration. As another example, the treatmentsession may comprise three administrations all given within one weeksuch as, e.g., given 1, 2 or 3 days after the preceding administration.As another example, the treatment session may comprise threeadministrations all given within two week such as, e.g., given 1, 2, 3,4 or 5 days after the preceding administration. As another example, thetreatment session may comprise four administrations all given within twoweek such as, e.g., given 1, 2, 3 or 4 days after the precedingadministration. As another example, the treatment session may comprisefour administrations all given within three week such as, e.g., given 1,2, 3, 4, 5 or 6 days after the preceding administration. As anotherexample, the treatment session may comprise five administrations allgiven within three week such as, e.g., given 1, 2, 3, 4 or 5 days afterthe preceding administration.

In certain embodiments, the interval session of non-treatment may bebetween one week and six months, for example between 2 weeks to 4 weeks,3 weeks to 4 weeks, 2 weeks to 6 weeks, 3 weeks to 6 weeks, 4 weeks to 6weeks, 5 weeks to 6 weeks, 2 weeks to 2 months, 3 weeks to 2 months, 4weeks to 2 months, 5 weeks to 2 months, 6 weeks to 2 months, 7 weeks to2 months, 2 months to 3 months, 2 months to 4 months, 3 months to 4months, 3 months to 5 months, 3 months to 5 months, 4 months to 5months, 1 week to 6 months, 2 weeks to 6 months, 3 weeks to 6 months, 4weeks to 6 months, 6 weeks to 6 months, 2 months to 6 months, 3 monthsto 6 months, 4 months to 6 months or 5 months to 6 months. In certainembodiments, the interval session of non-treatment may be 1 to 2 monthsin length, 1 to 3 months in length or 2 to 3 months in length.

In the treatments session, the administration of the active agent orpharmaceutical composition may be a single administration or repeatedadministration, for example the active agent or pharmaceuticalcomposition may be administered only once and then immediately followedby an interval, or it may be administered daily, or once every two,three, four, five or six days, or once weekly, once every two weeks,once every three weeks or once every four weeks. These frequencies areapplicable to any active agent, may be based on commonly used practicesin the art, and may finally be determined by physicians in clinicaltrials. Alternatively, the frequency of the repeated administration inthe treatment session could be adapted according to the nature of theactive agent, wherein for example, a small molecule may be administereddaily and an antibody may be administered once every 3 days. It shouldbe understood that when an agent is administered during a treatmentsession at a relatively low frequency, for example once per week duringa treatment session of one month, or once per month during a treatmentsession of six months, this treatment session is followed by anon-treatment interval session, the length of which is longer than theperiod between the repeated administrations during the treatment session(i.e. longer than one week or one month, respectively, in this example).The pause of one week or one month between the administrations duringthe treatment session in this example is not considered an intervalsession.

The lengths of the treatment session and the non-treatment or intervalsession may be adjusted to the frequency of the administration suchthat, for example, a frequency of administering the active agent onceevery 3 days may result in a treatment session of 6 or 9 days and aninterval session that is commenced accordingly.

If the treatment session consists of a single administration, the dosageregimen is determined by the length of the non-treatment interval, sothat a single administration is followed by a non-treatment interval of7, 8, 9, 10, 14, 18, 21, 24 or 28 days or longer before the nextsingle-administration treatment session. In particular, the dosageregimen consists of single administrations interspersed withnon-treatment intervals of non-treatment of 2, 3 or 4 weeks. Inaddition, the dosage regimen may consist of single administrationsinterspersed with non-treatment intervals of non-treatment of 2 to 4weeks, 2 to 3 weeks or 3 to 4 weeks.

If the treatment session consists of a multiple administrations, thedosage regimen is determined by the length of the non-treatmentinterval, so that multiple administrations given within one week isfollowed by a non-treatment interval of 7, 10, 14, 18, 21, 24 or 28 daysor longer before the next multiple-administration treatment session. Inparticular, the dosage regimen may consist of multiple administrationsgiven within one week interspersed with non-treatment intervals ofnon-treatment of 2 or 3 or 4 weeks. In addition, the dosage regimen mayconsist of multiple administrations given within one week interspersedwith intervals of non-treatment of 2 to 4 weeks, 2 to 3 weeks or 3 to 4weeks.

As another example, the dosage regimen may comprise multipleadministrations given within two weeks followed by a non-treatmentinterval of 2 weeks, 3 weeks or 1, 2, 3 or 4 months or longer before thenext multiple-administration treatment session. In particular, thedosage regimen may consist of multiple administrations given within twoweeks interspersed with intervals of non-treatment of 1, 2, 3 or 4months. In addition, the dosage regimen may consists of multipleadministrations given within two week interspersed with intervals ofnon-treatment of 1 to 2 months, 1 to 3 months, 1 to 4 months, 2 to 3months, 2 to 4 months or 3 to 4 months.

As another example, the dosage regimen may comprise multipleadministrations given within three week followed by 1, 2, 3, 4, 5 or 6months or longer of non-treatment before the nextmultiple-administration treatment session. In particular, the dosageregimen may consist of multiple administrations given within three weeksinterspersed with intervals of non-treatment of 1, 2, 3, 4, 5 or 6months. In addition, the dosage regimen may consists of multipleadministrations given within three weeks interspersed with intervals ofnon-treatment of 1 to 2 months, 1 to 3 months, 1 to 4 months, 1 to 5months, 1 to 6 months, 2 to 3 months, 2 to 4 months, 2 to 5 months, 2 to6 months, 3 to 4 months, 3 to 5 months, 3 to 6 months, 4 to 5 months, 4to 6 months or 5 to 6 months.

Of course, a flexible dosage regimen is envisioned that starts with acertain regimen and is replaced with another. For example, treatmentsessions, each one including 2 single administrations 3 days apart, withan interval of for example 1 week between the treatment sessions, couldbe replaced when considered appropriate by a dosage regimen includingtreatment sessions of single administrations separated by for example 2,3 or 4 weeks intervals. As another example, treatment sessions, each oneincluding 2 single administrations 7 days apart, with an interval of forexample 2 weeks between the treatment sessions, could be replaced whenconsidered appropriate by a dosage regimen including treatment sessionsof single administrations separated by for example 2, 3, 4, 5 or 6 weeksintervals. As another example, treatment sessions, each one including 3single administrations 3 days apart, with an interval of for example 2weeks between the treatment sessions, could be replaced when consideredappropriate by a dosage regimen including treatment sessions of singleadministrations separated by for example 2, 3, 4, 5 or 6 weeksintervals.

In any case, the dosage regimen, i.e. the length of the treatmentsession and the interval session, is calibrated such that the reductionin the level of immunosuppression, for example as measured by areduction in the level of systemic presence or activity of regulatory Tcells or the increase in the level of systemic presence or activity ofIFN-γ producing leukocytes in the individual, is transient.

The method, active agent or pharmaceutical composition according to thepresent invention may be for treating a disease, disorder or conditionof the CNS that is a neurodegenerative disease, disorder or condition.In aspects of this embodiment, the neurodegenerative disease, disorderor condition is Alzheimer's disease, amyotrophic lateral sclerosis,Parkinson's disease Huntington's disease, primary progressive multiplesclerosis; secondary progressive multiple sclerosis, corticobasaldegeneration, Rett syndrome, a tauopathy, a retinal degenerationdisorder; anterior ischemic optic neuropathy; glaucoma; uveitis;depression; trauma-associated stress or post-traumatic stress disorder,frontotemporal dementia, Lewy body dementias, mild cognitiveimpairments, posterior cortical atrophy, primary progressive aphasia orprogressive supranuclear palsy. In certain embodiments, the condition ofthe CNS is aged-related dementia. In certain aspects of this embodiment,the condition of the CNS is Alzheimer's disease, amyotrophic lateralsclerosis, Parkinson's disease Huntington's disease.

Tauopathies are a clinically, morphologically and biochemicallyheterogeneous class of neurodegenerative diseases characterized by apathological aggregation of tau protein in neurofibrillary orgliofibrillary tangles in the human brain. Tau is amicrotubule-associated protein (MAP) that binds to microtubules andpromotes their polymerization. It plays an important role in maintainingaxonal transport and neuronal integrity but has a physiological role indendrites, and it is expressed at low levels in glial cells. In atauopathy, tangles are formed by hyperphosphorylation of tau causing itto aggregate in an insoluble form. Non-limiting examples of tauopathiesinclude Alzheimer's disease, argyrophilic grain disease, chronictraumatic encephalopathy, corticobasal degeneration, dementiapugilistica, frontotemporal dementia, frontotemporal lobar degeneration,Hallervorden-Spatz disease, Huntington's disease, ganglioglioma,gangliocytoma, globular glial tauopathy, lead encephalopathy,lipofuscinosis, Lytico-Bodig disease (Parkinson-dementia complex ofGuam), meningioangiomatosis, Parkinsonism disease linked to chromosome17, Pick's disease, primary age-related tauopathy (PART), formerly knownas neurofibrillary tangle-only dementia (NFT-dementia), postencephaliticparkinsonism, progressive supranuclear palsy, subacute sclerosingpanencephalitis and tuberous sclerosis.

Retinal degeneration disorders are ones that result in the deteriorationof the retina due to the death of photoreceptor cells. There are severalcauses for retinal degeneration, including artery or vein occlusion,diabetic retinopathy, retrolental fibroplasia/retinopathy ofprematurity, or disease (usually hereditary). Symptoms include, withoutlimitation, impaired vision, night blindness, retinal detachment, lightsensitivity, glare sensitivity, tunnel vision, loss of depth perception,loss of contrast, night blindness, loss of central vision, loss ofperipheral vision and total loss of vision. Retinal degenerationdisorders include, without limitation, Age-Related Macular Degeneration(wet and dry), Retinitis Pigmentosa, Choroideremia, Cone-Rod RetinalDystrophy, Gyrate Atrophy, Juvenile Retinoschisis, Vitelliform MacularDystrophy (Best's Disease), Abetalipoproteinemia (Bassen-KornzweigDisease), Bardet-Biedl Syndrome, Blue Cone Monochromatism Disease,Dominant Drusen, Goldman-Favre Vitreoretinal Dystrophy (Enhanced S-coneSyndrome), Kearns-Sayre Syndrome, Laurence-Moon Syndrome, Leber'sCongenital Amaurosis, Leber's Refsum disease, Oguchi Disease,Peripapillary (pericentral) Choroidal Dystrophy, Pigment PatternDystrophy, Sorsby Macular Dystrophy, Stargardt's Disease, Stickler'sSyndrome, Usher Syndrome and Wagner's Vitreoretinal Dystrophy.

In certain embodiments, each one of the active agents described abovethat blocks one of the immune checkpoints selected from ICOS-B7RP1,V-domain Ig suppressor of T cell activation (VISTA), B7-CD28-likemolecule, CD40L-CD40, CD28-CD80, CD28-CD86, B7H3, B7H4, B7H7, BTLA-HVEM,CD137-CD137L, OX40L, CD27-CD70, STING, TIGIT and A2aR-Adenosine andindoleamine-2,3-dioxygenase (IDO)-L-tryptophan, such as an antibodyagainst one of the two partners of the immune checkpoint, is for use intreating either one of a neurodegenerative disease, disorder orcondition selected from Alzheimer's disease, amyotrophic lateralsclerosis, Parkinson's disease Huntington's disease, primary progressivemultiple sclerosis; secondary progressive multiple sclerosis,corticobasal degeneration, Rett syndrome, a retinal degenerationdisorder selected from the group consisting of age-related maculardegeneration and retinitis pigmentosa; anterior ischemic opticneuropathy; glaucoma; uveitis; depression; trauma-associated stress orpost-traumatic stress disorder, frontotemporal dementia, Lewy bodydementias, mild cognitive impairments, posterior cortical atrophy,primary progressive aphasia or progressive supranuclear palsy. Thetreatment of any one of these diseases comprising the use of any one ofthese active agents can be done according to the regiment describedabove.

The method, active agent and pharmaceutical composition according to thepresent invention may further be for treating an injury of the CNSselected from spinal cord injury, closed head injury, blunt trauma,penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebralischemia, optic nerve injury, myocardial infarction, organophosphatepoisoning and injury caused by tumor excision.

In certain embodiments, each one of the active agents described abovethat blocks one of the immune checkpoints selected from ICOS-B7RP1,V-domain Ig suppressor of T cell activation (VISTA), B7-CD28-likemolecule, CD40L-CD40, CD28-CD80, CD28-CD86, B7H3, B7H4, B7H7, BTLA-HVEM,CD137-CD137L, OX40L, CD27-CD70, STING, TIGIT and A2aR-Adenosine andindoleamine-2,3-dioxygenase (IDO)-L-tryptophan, such as an antibodyagainst one of the two partners of the immune checkpoint, is for use intreating an injury of the CNS selected from spinal cord injury, closedhead injury, blunt trauma, penetrating trauma, hemorrhagic stroke,ischemic stroke, cerebral ischemia, optic nerve injury, myocardialinfarction, organophosphate poisoning and injury caused by tumorexcision. The treatment of any one of these injuries comprising the useof any one of these active agents can be done according to the regimentdescribed above.

As stated above, the inventors have found that the present inventionimproves the cognitive function in mice that emulates Alzheimer'sdisease. Thus, the method, active agent and pharmaceutical compositionmay be for use in improving CNS motor and/or cognitive function, forexample for use in alleviating age-associated loss of cognitivefunction, which may occur in individuals free of a diagnosed disease, aswell as in people suffering from neurodegenerative disease. Furthermore,the method, active agent and pharmaceutical composition may be for usein alleviating loss of cognitive function resulting from acute stress ortraumatic episode. The cognitive function mentioned herein above maycomprise learning, memory or both.

It should be emphasized, that the improvement of cognitive function inmice that emulates Alzheimer's disease (5XFAD AD-Tg mice) were observedand characterized by the inventors in various stages of diseasemanifestation; both early and late progressive stages of diseasepathology could be mitigated by the treatment. 5XFAD AD-Tg mice begin todisplay cerebral plaque pathology at the ages of 2.5 months andcognitive deficits at the ages of 5 months (Oakley et al, 2006). Ofnote, while in Example 2 below the inventors describe the therapeuticeffect in 5XFAD mice at 6 months of age, in Example 5 they characterizethe therapeutic effect in 5XFAD mice at 11 and 12 months of age—anextremely progressive stage of amyloid beta plaque deposition andcognitive deficits in this model. It is therefore expected that theproposed invention would be of relevance to patients of different stagesof disease progression, such as Stage 1—Mild/Early (lasts 2-4 years);Stage 2—Moderate/Middle (lasts 2-10 years); and Stage 3—Severe/Late(lasts 1-3+ years).

The term “CNS function” as used herein refers, inter alia, to receivingand processing sensory information, thinking, learning, memorizing,perceiving, producing and understanding language, controlling motorfunction and auditory and visual responses, maintaining balance andequilibrium, movement coordination, the conduction of sensoryinformation and controlling such autonomic functions as breathing, heartrate, and digestion.

The terms “cognition”, “cognitive function” and “cognitive performance”are used herein interchangeably and are related to any mental process orstate that involves but is not limited to learning, memory, creation ofimagery, thinking, awareness, reasoning, spatial ability, speech andlanguage skills, language acquisition and capacity for judgmentattention. Cognition is formed in multiple areas of the brain such ashippocampus, cortex and other brain structures. However, it is assumedthat long term memories are stored at least in part in the cortex and itis known that sensory information is acquired, consolidated andretrieved by a specific cortical structure, the gustatory cortex, whichresides within the insular cortex.

In humans, cognitive function may be measured by any know method, forexample and without limitation, by the clinical global impression ofchange scale (CIBIC-plus scale); the Mini Mental State Exam (MMSE); theNeuropsychiatric Inventory (NPI); the Clinical Dementia Rating Scale(CDR); the Cambridge Neuropsychological Test Automated Battery (CANTAB)or the Sandoz Clinical Assessment-Geriatric (SCAG). Cognitive functionmay also be measured indirectly using imaging techniques such asPositron Emission Tomography (PET), functional magnetic resonanceimaging (fMRI), Single Photon Emission Computed Tomography (SPECT), orany other imaging technique that allows one to measure brain function.

An improvement of one or more of the processes affecting the cognitionin a patient will signify an improvement of the cognitive function insaid patient, thus in certain embodiments improving cognition comprisesimproving learning, plasticity, and/or long term memory. The terms“improving” and “enhancing” may be used interchangeably.

The term “learning” relates to acquiring or gaining new, or modifyingand reinforcing, existing knowledge, behaviors, skills, values, orpreferences.

The term “plasticity” relates to synaptic plasticity, brain plasticityor neuroplasticity associated with the ability of the brain to changewith learning, and to change the already acquired memory. One measurableparameter reflecting plasticity is memory extinction.

The term “memory” relates to the process in which information isencoded, stored, and retrieved. Memory has three distinguishablecategories: sensory memory, short-term memory, and long-term memory.

The term “long term memory” is the ability to keep information for along or unlimited period of time. Long term memory comprises two majordivisions: explicit memory (declarative memory) and implicit memory(non-declarative memory). Long term memory is achieved by memoryconsolidation which is a category of processes that stabilize a memorytrace after its initial acquisition. Consolidation is distinguished intotwo specific processes, synaptic consolidation, which occurs within thefirst few hours after learning, and system consolidation, wherehippocampus-dependent memories become independent of the hippocampusover a period of weeks to years.

The embodiments above that describe different features of thepharmaceutical composition of the present invention are relevant alsofor the method of the invention, because the method employs the samepharmaceutical composition.

In yet another aspect, the present invention provides methods forreducing Aβ-plaque burden in a patient diagnosed with Alzheimer'sdisease, comprising administering to said patient an active agent orpharmaceutical composition as defined herein above that causes reductionof the level of systemic immunosuppression by release of a restraintimposed on the immune system by one or more immune checkpoints.

In still another aspect, the present invention provides a method forreducing hippocampal gliosis in a patient diagnosed with Alzheimer'sdisease, comprising administering to said patient an active agent orpharmaceutical composition as defined herein above that causes reductionof the level of systemic immunosuppression by release of a restraintimposed on the immune system by one or more immune checkpoints.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The carrier(s) mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof.

The following exemplification of carriers, modes of administration,dosage forms, etc., are listed as known possibilities from which thecarriers, modes of administration, dosage forms, etc., may be selectedfor use with the present invention. Those of ordinary skill in the artwill understand, however, that any given formulation and mode ofadministration selected should first be tested to determine that itachieves the desired results.

Methods of administration include, but are not limited to, parenteral,e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal(e.g., oral, intranasal, buccal, vaginal, rectal, intraocular),intrathecal, topical and intradermal routes. Administration can besystemic or local.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the active agent is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; and a glidant, such as colloidal silicondioxide.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compositions may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen free water, before use.

The compositions may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The determination of the doses of the active ingredient to be used forhuman use is based on commonly used practices in the art, and will befinally determined by physicians in clinical trials. An expectedapproximate equivalent dose for administration to a human can becalculated based on the in vivo experimental evidence disclosed hereinbelow, using known formulas (e.g. Reagan-Show et al. (2007) Dosetranslation from animal to human studies revisited. The FASEB Journal22:659-661). According to this paradigm, the adult human equivalent dose(mg/kg body weight) equals a dose given to a mouse (mg/kg body weight)multiplied with 0.081.

Aspects of the present specification can also be described as follows:

-   1. A method of treating a treating a disease, disorder, condition or    injury of the Central Nervous System to an individual in need    thereof, the method comprising administering to the individual a    composition comprising an active agent that causes reduction of the    level of systemic immunosuppression by release of a restraint    imposed on the immune system by one or more immune checkpoints,    wherein the composition is administered by a dosage regime    comprising at least one course of therapy, each course of therapy    comprising in sequence a treatment session where the composition is    administered to the individual followed by a non-treatment session    where the composition is not administered to the individual, wherein    the non-treatment period is longer than the treatment session;    wherein, if administration of the composition during the treatment    session is a repeated administration, the non-treatment period is    longer than the period between repeated administrations during the    treatment session; wherein administration of the composition    transiently reduces levels of systemic immunosuppression and    increases choroid plexus gateway activity in facilitating selective    recruitment of immune cells into the central nervous system, thereby    treating the individual.-   2. Use of an active agent that causes reduction of the level of    systemic immunosuppression by release of a restraint imposed on the    immune system by one or more immune checkpoints in the treatment of    a disease, disorder, condition or injury of the Central Nervous    System.-   3. Use of an active agent that causes reduction of the level of    systemic immunosuppression by release of a restraint imposed on the    immune system by one or more immune checkpoints in the manufacture    of a medicament for the treatment of a disease, disorder, condition    or injury of the Central Nervous System.-   4. The method according to embodiment 1 or the use according to    embodiments 2 or 3, wherein the active agent is an antibody, an    antibody mimetic, an aptamer, a small molecule, a Neem leaf    glycoprotein, a sCTLA-4, a silencing molecule, an OK-432, a    combination of IL-12 and anti-CTLA-4, or any combination thereof.-   5. The method according to embodiment 4 or the use according to    embodiment 4, wherein the antibody is a polyclonal antibody or a    monoclonal antibody.-   6. The method according to embodiments 4 or 5 or the use according    to embodiments 4 or 5, wherein the antibody is a dimer, a multimer,    a multispecific antibody, a recombinant antibody, a chimeric    antibody, bi-functional antibody, a cell-associated antibody like an    Ig receptor, a linear antibody, a diabody, a minibody or a nanobody.-   7. The method according to any one of embodiments 4-6 or the use    according to any one of embodiments 4-6, wherein the antibody is a    human antibody or a humanized antibody.-   8. The method according to any one of embodiments 4-7 or the use    according to any one of embodiments 4-7, wherein the antibody is an    antagonistic antibody or an agonistic antibody.-   9. The method according to any one of embodiments 4-8 or the use    according to any one of embodiments 4-8, wherein the antibody is a    neutralizing antibody.-   10. The method according to any one of embodiments 4-9 or the use    according to any one of embodiments 4-9, wherein the antibody is a    full-length immunoglobulin molecule or an immunologically active    fragment.-   11. The method according to embodiment 10 or the use according to    embodiment 10, wherein the immunologically active fragment is a    single domain antibody (sdAb), a single-chain variable fragment    (scFv), a Fab fragment, a F(ab′)2 fragment, a Fc fragment, a Fd    fragment, a Fv fragment.-   12. The method according to any one of embodiments 4-11 or the use    according to any one of embodiments 4-11, wherein the antibody is an    anti-PD-1, an anti-PD-L1, an anti-PD-L2, an anti-CTLA-4, an    anti-CD80, an anti-CD86, an anti-B7RP1, an anti-B7-H3, an    anti-B7-H4, an anti-B7-H7, an anti-BTLA, an anti-HVEM, an    anti-CD-27, an anti-CD40, an anti-CD40L, an anti-CD70, an anti-CD80,    an anti-CD86, an anti-CD137, an anti-CD137L, an anti-OX40, an    anti-OX40L, an anti-TIM-3, an anti-Galectin9, an anti-KIR, an    anti-LAG-3, an anti-ICOS, an anti-VISTA, an anti-STING, an    anti-TIGIT, anti-GITR or any combination thereof.-   13. The method according to embodiment 4 or the use according to    embodiment 4, wherein the antibody mimetic is an affibody molecule,    an affilin, an affimer, an affitin, an alphabody, an anticalin, an    avimer, a DARPin, a fynomer, a Kunitz domain peptide, or a monobody.-   14. The method according to embodiment 4 or the use according to    embodiment 4, wherein the aptamer is a DNA aptamer, a RNA aptamer, a    XNA aptamer or a peptide aptamer.-   15. The method according to embodiment 4 or the use according to    embodiment 4, wherein the small molecule is a p300 inhibitor, a    Sunitinib, a Polyoxometalate-1, an α,β-methyleneadenosine    5′-diphosphate, an arsenic trioxide, a GX15-070, a retinoic acid    antagonist, a CCR4 antagonist, an adenosine receptor antagonist, an    adenosine A1 receptor antagonist; an adenosine A2a receptor, an    adenosine A2b receptor antagonist, an A3 receptor antagonist, an    antagonist of indoleamine-2,3-dioxygenase or an HIF-1 regulator.-   16. The method according to any one of embodiments 1-15 or the use    according to any one of embodiments 4-15, wherein the administration    of the composition during the treatment session is a single    administration.-   17. The method according to any one of embodiments 1-15 or the use    according to any one of embodiments 4-15, wherein the administration    of the composition during the treatment session is a repeated    administration.-   18. The method according to embodiment 17 or the use according to    embodiment 17, wherein the repeated administration occurs once every    day, once every two days, once every three days, once every four    days, once every five days or once every six days.-   19. The method according to embodiment 17 or the use according to    embodiment 17, wherein the repeated administration occurs once    weekly or once every two weeks, once every three weeks or once every    four weeks.-   20. The method according to any one of embodiments 1-19 or the use    according to any one of embodiments 1-19, wherein the treatment    session is from 1 day to four weeks.-   21. The method according to embodiment 20 or the use according to    embodiment 20, wherein the treatment session is from 3 days to four    weeks.-   22. The method according to embodiment 21 or the use according to    embodiment 21, wherein the treatment session is from one week to    four weeks.-   23. The method according to any one of embodiments 1-22 or the use    according to any one of embodiments 1-22, wherein the non-treatment    period is from one week to six months.-   24. The method according to embodiment 23 or the use according to    embodiment 23, wherein the non-treatment period is from two weeks to    six months.-   25. The method according to embodiment 24 or the use according to    embodiment 24, wherein the non-treatment period is from three weeks    to six months.-   26. The method according to embodiment 25 or the use according to    embodiment 25, wherein the non-treatment period is from one month to    three months.-   27. The method according to embodiment 26 or the use according to    embodiment 26, wherein the non-treatment period is from one month to    two months.-   28. The method according to any one of embodiments 1-27 or the use    according to any one of embodiments 1-27, wherein the transient    reduction in the level of systemic immunosuppression is associated    with an increase in a systemic presence or activity of    IFNγ-producing leukocytes and/or an increase in a systemic presence    or activity of an IFNγ cytokine.-   29. The method according to any one of embodiments 1-28 or the use    according to any one of embodiments 1-28, wherein the transient    reduction in the level of systemic immunosuppression is associated    with an increase in a systemic presence or activity of effector T    cells.-   30. The method according to any one of embodiments 1-29 or the use    according to any one of embodiments 1-29, wherein the transient    reduction in the level of systemic immunosuppression is associated    with a decrease in a systemic presence or activity of regulatory T    cells and/or a decrease in a systemic presence of an IL-10 cytokine.-   31. The method according to any one of embodiments 1-30 or the use    according to any one of embodiments 1-30, wherein the transient    reduction in the level of systemic immunosuppression is associated    with a decrease in a systemic presence or myeloid-derived suppressor    cells (MDSCs).-   32. The method according to any one of embodiments 1-31 or the use    according to any one of embodiments 1-31, wherein the transient    reduction in the level of systemic immunosuppression occurs by    release of a restraint imposed on the immune system by one or more    immune checkpoints.-   33. The method according to embodiment 32 or the use according to    embodiment 32, wherein administration of the composition blocks the    one or more immune checkpoints, thereby causing the transient    reduction in the level of systemic immunosuppression.-   34. The method according to embodiment 33 or the use according to    embodiment 33, wherein the one or more immune checkpoints includes a    PD-1-PD-L1, a PD-1-PD-L2, a CD28-CD80, a CD28-CD86, a CTLA-4-CD80, a    CTLA-4-CD86, an ICOS-B7RP1, a B7H3, a B7H4, a B7H7, a B7-CD28-like    molecule, a BTLA-HVEM, a KIR-MHC class I or II,a LAG3-MHC class I or    II, a CD137-CD137L, an OX40-OX40L, a CD27-CD70, a CD40L-CD40, a    TIM3-GAL9, a V-domain Ig suppressor of T cell activation (VISTA), a    STimulator of INterferon Genes (STING), a T cell immunoglobulin and    immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), a    glucocorticoid-induced tumor necrosis factor receptor related    protein (GITR), an A2aR-Adenosine or an indoleamine-2,3-dioxygenase    (IDO)-L-tryptophan.-   35. The method according to any one of embodiments 1-34 or the use    according to any one of embodiments 1-34, wherein the administration    of the composition during the treatment session is maintained at    least until a systemic presence or activity of IFNγ-producing    leukocytes and/or an IFNγ cytokine rises above a reference, at which    point the administration is stopped, and the non-treatment period is    maintained as long as the systemic presence or activity of    IFNγ-producing leukocytes and/or an IFNγ cytokine is above the    reference, wherein the reference includes a) a level of a systemic    presence or activity of IFNγ-producing leukocytes and/or an IFNγ    cytokine measured in the most recent blood sample obtained from the    individual before the administering; or b) a level of a systemic    presence or activity of IFNγ-producing leukocytes and/or an IFNγ    cytokine characteristic of a population of individuals afflicted    with the disease, disorder, condition or injury of the Central    Nervous System.-   36. The method according to any one of embodiments 1-35 or the use    according to any one of embodiments 1-35, wherein a cerebral level    of soluble amyloid beta peptide is reduced in the individual, a    cerebral amyloid beta (Aβ) plaque burden is reduced or cleared in    the individual, a hippocampal gliosis is reduced in the individual,    a cerebral level of a pro-inflammatory cytokine is reduced in the    individual, a brain inflammation is decreased in the individual    and/or a cognitive function is improved in the individual.-   37. The method according to embodiment 36 or the use according to    embodiment 36, wherein the improved cognitive function is learning,    memory, creation of imagery, plasticity, thinking, awareness,    reasoning, spatial ability, speech and language skills, language    acquisition, capacity for judgment attention or any combination    thereof.-   38. The method according to any one of embodiments 1-37 or the use    according to any one of embodiments 1-37, wherein the immune cells    include monocytes, macrophages, or T cells.-   39. The method according to embodiment 38 or the use according to    embodiment 38, wherein the T cells include regulatory T cells.-   40. The method according to any one of embodiments 1-39 or the use    according to any one of embodiments 1-39, wherein the disease,    disorder, condition or injury of the Central Nervous System is    Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's    disease Huntington's disease, primary progressive multiple    sclerosis, secondary progressive multiple sclerosis, corticobasal    degeneration, Rett syndrome, anterior ischemic optic neuropathy,    glaucoma, uveitis, depression, trauma-associated stress or    post-traumatic stress disorder, frontotemporal dementia, Lewy body    dementias, mild cognitive impairments, posterior cortical atrophy,    primary progressive aphasia or progressive supranuclear palsy-   41. The method according to any one of embodiments 1-39 or the use    according to any one of embodiments 1-39, wherein the disease,    disorder, condition or injury of the Central Nervous System is a    tauopathy.-   42. The method according to embodiment 41 or the use according to    embodiment 41, wherein the tauopathy is Alzheimer's disease,    argyrophilic grain disease, chronic traumatic encephalopathy,    corticobasal degeneration, dementia pugilistica, frontotemporal    dementia, frontotemporal lobar degeneration, Hallervorden-Spatz    disease, Huntington's disease, ganglioglioma, gangliocytoma,    globular glial tauopathy, lead encephalopathy, lipofuscinosis,    Lytico-Bodig disease (Parkinson-dementia complex of Guam),    meningioangiomatosis, Parkinsonism disease linked to chromosome 17,    Pick's disease, primary age-related tauopathy (PART), formerly known    as neurofibrillary tangle-only dementia (NFT-dementia),    postencephalitic parkinsonism, progressive supranuclear palsy,    subacute sclerosing panencephalitis or tuberous sclerosis.-   43. The method according to any one of embodiments 1-39 or the use    according to any one of embodiments 1-39, wherein the disease,    disorder, condition or injury of the Central Nervous System is a    retinal degeneration disorder.-   44. The method according to embodiment 43 or the use according to    embodiment 43, wherein the retinal degeneration disorder is wet    Age-Related Macular Degeneration, dry Age-Related Macular    Degeneration, Retinitis Pigmentosa, Choroideremia, Cone-Rod Retinal    Dystrophy, Gyrate Atrophy, Juvenile Retinoschisis, Vitelliform    Macular Dystrophy (Best's Disease), Abetalipoproteinemia    (Bassen-Kornzweig Disease), Bardet-Biedl Syndrome, Blue Cone    Monochromatism Disease, Dominant Drusen, Goldman-Favre Vitreoretinal    Dystrophy (Enhanced S-cone Syndrome), Kearns-Sayre Syndrome,    Laurence-Moon Syndrome, Leber's Congenital Amaurosis, Leber's Refsum    disease, Oguchi Disease, Peripapillary (pericentral) Choroidal    Dystrophy, Pigment Pattern Dystrophy, Sorsby Macular Dystrophy,    Stargardt's Disease, Stickler's Syndrome, Usher Syndrome or Wagner's    Vitreoretinal Dystrophy.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the active agents,pharmaceutical compositions, or methods and uses disclosed herein.

Materials and Methods

Animals. 5XFAD transgenic mice (Tg6799) that co-overexpress familial ADmutant forms of human APP (the Swedish mutation, K670N/M671L; theFlorida mutation, 1716V; and the London mutation, V7171) and PS1(M146L/L286V) transgenes under transcriptional control of theneuron-specific mouse Thy-1 promoter (Oakley et al, 2006), and AD doubletransgenic B6.Cg-Tg (APPswe, PSEN1dE9) 85Dbo/J mice (Borchelt et al,1997) were purchased from The Jackson Laboratory. Genotyping wasperformed by PCR analysis of tail DNA, as previously described (Oakleyet al, 2006). Heterozygous mutant cx3cr1^(GFP/+) mice (Jung et al, 2000)(B6.129P-cx3cr1^(tm1Litt)/J, in which one of the CX₃CR1 chemokinereceptor alleles was replaced with a gene encoding GFP) were used asdonors for BM chimeras. Foxp3.LuciDTR mice (Suffner et al, 2010) werebred with 5XFAD mice to enable conditional depletion of Foxp3⁺ Tregs.Animals were bred and maintained by the Animal Breeding Center of theWeizmann Institute of Science. All experiments detailed herein compliedwith the regulations formulated by the Institutional Animal Care and UseCommittee (IACUC) of the Weizmann Institute of Science.

RNA purification, cDNA synthesis, and quantitative real-time PCRanalysis. Total RNA of the hippocampal dentate gyrus (DG) was extractedwith TRI Reagent (Molecular Research Center) and purified from thelysates using an RNeasy Kit (Qiagen). Total RNA of the choroid plexuswas extracted using an RNA MicroPrep Kit (Zymo Research). mRNA (1 μg)was converted into cDNA using a High Capacity cDNA Reverse TranscriptionKit (Applied Biosystems). The expression of specific mRNAs was assayedusing fluorescence-based quantitative real-time PCR (RT-qPCR). RT-qPCRreactions were performed using Fast-SYBR PCR Master Mix (AppliedBiosystems). Quantification reactions were performed in triplicate foreach sample using the standard curve method. Peptidylprolyl isomerase A(ppia) was chosen as a reference (housekeeping) gene. The amplificationcycles were 95° C. for 5 s, 60° C. for 20 s, and 72° C. for 15 s. At theend of the assay, a melting curve was constructed to evaluate thespecificity of the reaction. For ifn-γ and ppia gene analysis, the cDNAwas pre-amplified in 14 PCR cycles with non-random PCR primers, therebyincreasing the sensitivity of the subsequent real-time PCR analysis,according to the manufacturer's protocol (PreAmp Master Mix Kit; AppliedBiosystems). mRNA expression was determined using TaqMan RT-qPCR,according to the manufacturer's instructions (Applied Biosystems). AllRT-qPCR reactions were performed and analyzed using StepOne softwareV2.2.2 (Applied Biosystems). The following TaqMan Assays-on-Demand™probes were used: Mm02342430_g1 (ppia) and Mm01168134_m1 (ifn-γ).

For all other genes examined, the following primers were used:

ppia forward (SEQ ID NO: 33) 5′-AGCATACAGGTCCTGGCATCTTGT-3′ and reverse(SEQ ID NO: 34) 5′-CAAAGACCACATGCTTGCCATCCA-3′; icam1 forward(SEQ ID NO: 35) 5′-AGATCACATTCACGGTGCTGGCTA-3′ and reverse(SEQ ID NO: 36) 5′-AGCTTTGGGATGGTAGCTGGAAGA-3′; vcam1 forward(SEQ ID NO: 37) 5′-TGTGAAGGGATTAACGAGGCTGGA-3′ and reverse(SEQ ID NO: 38) 5′-CCATGTTTCGGGCACATTTCCACA-3′; cxcl10 forward(SEQ ID NO: 39) 5′-AACTGCATCCATATCGATGAC-3′ and  reverse (SEQ ID NO: 40)5′-GTGGCAATGATCTCAACAC-3′; ccl2 forward (SEQ ID NO: 41)5′-CATCCACGTGTTGGCTCA-3′ and reverse (SEQ ID NO: 42)5′-GATCATCTTGCTGGTGAATGAGT-3′; tnf-γ forward (SEQ ID NO: 43)5′-GCCTCTTCTCATTCCTGCTT-3′ reverse (SEQ ID NO: 44)CTCCTCCACTTGGTGGTTTG-3′; il-1β forward (SEQ ID NO: 45)5′-CCAAAAGATGAAGGGCTGCTT-3′ and reverse (SEQ ID NO: 46)5′-TGCTGCTGCGAGATTTGAAG-3′; il-12p40 forward (SEQ ID NO: 47)5′-GAAGTTCAACATCAAGAGCA-3′ and reverse (SEQ ID NO: 48)5′-CATAGTCCCTTTGGTCCAG-3′; il-10 forward (SEQ ID NO: 49)5′-TGAATTCCCTGGGTGAGAAGCTGA-3′ and reverse (SEQ ID NO: 50)5′-TGGCCTTGTAGACACCTTGGTCTT-3′; tgfβ forward (SEQ ID NO: 51)5′-AATTGCTGCCTTCGCCCTCTTTAC-3′ and reverse (SEQ ID NO: 52)5′-TGTACAGGCTGAGGACTTTGGTGT-3′; igf-1 forward (SEQ ID NO: 53)5′-CCGGACCAGAGACCCTTTG and reverse (SEQ ID NO: 54)5′-CCTGTGGGCTTGTTGAAGTAAAA-3′; bdnf forward (SEQ ID NO: 55)5′-GATGCTCAGCAGTCAAGTGCCTTT-3′ and reverse (SEQ ID NO: 56)5′-GACATGTTTGCGGCATCCAGGTAA-3′;

Immunohistochemistry. Tissue processing and immunohistochemistry wereperformed on paraffin embedded sectioned mouse (6 μm thick) and human(10 μm thick) brains. For human ICAM-1 staining, primary mouse anti-ICAM(1:20 Abcam; ab2213) antibody was used. Slides were incubated for 10 minwith 3% H2O2, and a secondary biotin-conjugated anti-mouse antibody wasused, followed by biotin/avidin amplification with Vectastain ABC kit(Vector Laboratories). Subsequently, 3,3′-diaminobenzidine (DABsubstrate) (Zytomed kit) was applied; slides were dehydrated and mountedwith xylene-based mounting solution. For tissue stainings, mice weretranscardially perfused with PBS prior to tissue excision and fixation.CP tissues were isolated under a dissecting microscope (Stemi DV4;Zeiss) from the lateral, third, and fourth ventricles of the brain. Forwhole mount CP staining, tissues were fixated with 2.5% paraformaldehyde(PFA) for 1 hour at 4° C., and subsequently transferred to PBScontaining 0.05% sodium azide. Prior to staining, the dissected tissueswere washed with PBS and blocked (20% horse serum, 0.3% Triton X-100,and PBS) for 1 h at room temperature. Whole mount staining with primaryantibodies (in PBS containing 2% horse serum and 0.3% Triton X-100), orsecondary antibodies, was performed for 1 h at room temperature. Eachstep was followed by three washes in PBS. The tissues were applied toslides, mounted with Immu-mount (9990402, from Thermo Scientific), andsealed with cover-slips. For staining of sectioned brains, two differenttissue preparation protocols (paraffin embedded or microtomedfree-floating sections) were applied, as previously described (Baruch etal, 2013; Kunis et al, 2013). The following primary antibodies wereused: mouse anti-Aβ (1:300, Covance, # SIG-39320); rabbit anti-GFP(1:100, MBL, #598); rat anti-CD68 (1:300, eBioscience, #14-0681); ratanti-ICAM-1 (1:200, Abcam, # AB2213); goat anti-GFP (1:100, Abcam, #ab6658); rabbit anti-IBA-1 (1:300, Wako, #019-19741); goat anti-IL-10(1:20, R&D systems, # AF519); rat anti-Foxp3 (1:20, eBioscience,#13-5773-80); rabbit anti-CD3 (1:500, Dako, # IS503);; mouse anti-ZO-1,mouse anti-E-Cahedrin, and rabbit anti-Claudin-1 (all 1:100, Invitrogen,#33-9100, #33-4000, #51-9000); rabbit anti-GFAP (1:200, Dako, # Z0334).Secondary antibodies included: Cy2/Cy3/Cy5-conjugated donkeyanti-mouse/goat/rabbit/rat antibodies (1:200; all from JacksonImmunoresearch). The slides were exposed to Hoechst nuclear staining(1:4000; Invitrogen Probes) for 1 min. Two negative controls wereroutinely used in immunostaining procedures, staining with isotypecontrol antibody followed by secondary antibody, or staining withsecondary antibody alone. For Foxp3 intracellular staining, antigenretrieval from paraffin-embedded slides was performed using RetreivagenKit (#550524, #550527; BD Pharmingen™) Microscopic analysis, wasperformed using a fluorescence microscope (E800; Nikon) orlaser-scanning confocal microscope (Carl Zeiss, Inc.). The fluorescencemicroscope was equipped with a digital camera (DXM 1200F; Nikon), andwith either a 20×NA 0.50 or 40×NA 0.75 objective lens (Plan Fluor;Nikon). The confocal microscope was equipped with LSM 510 laser scanningcapacity (three lasers: Ar 488, HeNe 543, and HeNe 633). Recordings weremade on postfixed tissues using acquisition software (NIS-Elements, F3[Nikon] or LSM [Carl Zeiss, Inc.]). For quantification of stainingintensity, total cell and background staining was measured using ImageJsoftware (NIH), and intensity of specific staining was calculated, aspreviously described (Burgess et al, 2010). Images were cropped, merged,and optimized using Photoshop CS6 13.0 (Adobe), and were arranged usingIllustrator CS5 15.1 (Adobe).

Paraffin embedded sections of human CP. Human brain sections of youngand aged postmortem non-CNS-disease individuals, as well as AD patients,were obtained from the Oxford Brain Bank (formerly known as the ThomasWillis Oxford Brain Collection (TWOBC)) with appropriate consent andEthics Committee approval (TW220). The experiments involving thesesections were approved by the Weizmann Institute of Science BioethicsCommittee.

Flow cytometry, sample preparation and analysis. Mice weretranscardially perfused with PBS, and tissues were treated as previouslydescribed (Baruch et al, 2013). Brains were dissected and the differentbrain regions were removed under a dissecting microscope (Stemi DV4;Zeiss) in PBS, and tissues were dissociated using the gentleMACS™dissociator (Miltenyi Biotec). Choroid plexus tissues were isolated fromthe lateral, third and fourth ventricles of the brain, incubated at 37°C. for 45 min in PBS (with Ca²⁺/Mg²⁺) containing 400 U/ml collagenasetype IV (Worthington Biochemical Corporation), and then manuallyhomogenized by pipetting. Spleens were mashed with the plunger of asyringe and treated with ACK (ammonium chloride potassium) lysing bufferto remove erythrocytes. In all cases, samples were stained according tothe manufacturers' protocols. All samples were filtered through a 70 μmnylon mesh, and blocked with anti-Fc CD16/32 (1:100; BD Biosciences).For intracellular staining of IFN-γ, the cells were incubated withpara-methoxyamphetamine (10 ng/ml; Sigma-Aldrich) and ionomycin (250ng/ml; Sigma-Aldrich) for 6 h, and Brefeldin-A (10 μg/ml; Sigma-Aldrich)was added for the last 4 h. Intracellular labeling of cytokines was donewith BD Cytofix/Cytoperm™ Plus fixation/permeabilization kit (cat. no.555028). For Treg staining, an eBioscience FoxP3 staining buffer set(cat. no. 00-5523-00) was used. The following fluorochrome-labeledmonoclonal antibodies were purchased from BD Pharmingen, BioLegend, R&DSystems, or eBiosciences, and used according to the manufacturers'protocols: PE or Alexa Fluor 450-conjugated anti-CD4; PE-conjugatedanti-CD25; PerCP-Cy5.5-conjugated anti-CD45; FITC-conjugated anti-TCRI3;APC-conjugated anti-IFN-γ; APC-conjugated anti-FoxP3;Brilliant-violet-conjugated anti-CD45. Cells were analyzed on an LSRIIcytometer (BD Biosciences) using FlowJo software. In each experiment,relevant negative control groups, positive controls, and single stainedsamples for each tissue were used to identify the populations ofinterest and to exclude other populations.

Preparation of BM chimeras. BM chimeras were prepared as previouslydescribed (Shechter et al, 2009; Shechter et al, 2013). In brief,gender-matched recipient mice were subjected to lethal whole-bodyirradiation (950 rad) while shielding the head (Shechter et al, 2009).The mice were then injected intravenously with 5×10⁶ BM cells fromCX3CR1^(GFP/+) donors. Mice were left for 8-10 weeks after BMtransplantation to enable reconstitution of the hematopoietic lineage,prior to their use in experiments. The percentage of chimerism wasdetermined by FACS analysis of blood samples according to percentages ofGFP expressing cells out of circulating monocytes (CD11b⁺). In thishead-shielded model, an average of 60% chimerism was achieved, andCNS-infiltrating GFP⁺ myeloid cells were verified to beCD45^(high)/CD11b^(high), representing monocyte-derived macrophages andnot microglia (Shechter et al, 2013).

Morris Water Maze. Mice were given three trials per day, for 4consecutive days, to learn to find a hidden platform located 1.5 cmbelow the water surface in a pool (1.1 m in diameter). The watertemperature was kept between 21-22° C. Water was made opaque with milkpowder. Within the testing room, only distal visual shape and objectcues were available to the mice to aid in location of the submergedplatform. The escape latency, i.e., the time required to find and climbonto the platform, was recorded for up to 60 s. Each mouse was allowedto remain on the platform for 15 s and was then removed from the maze toits home cage. If the mouse did not find the platform within 60 s, itwas manually placed on the platform and returned to its home cage after15 s. The inter-trial interval for each mouse was 10 min. On day 5, theplatform was removed, and mice were given a single trial lasting 60 swithout available escape. On days 6 and 7, the platform was placed inthe quadrant opposite the original training quadrant, and the mouse wasretrained for three sessions each day. Data were recorded using theEthoVision V7.1 automated tracking system (Noldus InformationTechnology). Statistical analysis was performed using analysis ofvariance (ANOVA) and the Bonferroni post-hoc test. All MWM testing wasperformed between 10 a.m. and 5 p.m. during the lights-off phase.

Radial Arm Water Maze. The radial-arm water maze (RAWM) was used to testspatial learning and memory, as was previously described in detail(Alamed et al, 2006). Briefly, six stainless steel inserts were placedin the tank, forming six swim arms radiating from an open central area.The escape platform was located at the end of one arm (the goal arm),1.5 cm below the water surface, in a pool 1.1 m in diameter. The watertemperature was kept between 21-22° C. Water was made opaque with milkpowder. Within the testing room, only distal visual shape and objectcues were available to the mice to aid in location of the submergedplatform. The goal arm location remained constant for a given mouse. Onday 1, mice were trained for 15 trials (spaced over 3 h), with trialsalternating between a visible and hidden platform, and the last 4 trailswith hidden platform only. On day 2, mice were trained for 15 trialswith the hidden platform. Entry into an incorrect arm, or failure toselect an arm within 15 sec, was scored as an error. Spatial learningand memory were measured by counting the number of arm entry errors orthe escape latency of the mice on each trial. Training data wereanalyzed as the mean errors or escape latency, for training blocks ofthree consecutive trials.

GA administration. Each mouse was subcutaneously (s.c.) injected with atotal dose of 100 μg of GA (batch no. P53640; Teva PharmaceuticalIndustries, Petah Tiqva, Israel) dissolved in 200 μl of PBS. Mice wereeither injected according to a weekly-GA regimen (Butovsky et al, 2006),or daily-GA administration (FIG. 8 and FIG. 16 ). Mice were euthanizedeither 1 week after the last GA injection, or 1 month after treatment,as indicated for each experiment.

Conditional ablation of Treg. Diphtheria toxin (DTx; 8 ng/g body weight;Sigma) was injected intraperitoneally (i.p.) daily for 4 consecutivedays to Foxp3.LuciDTR mice (Suffner et al, 2010). The efficiency of DTxwas confirmed by flow cytometry analysis of immune cells in the bloodand spleen, achieving almost complete (>99%) depletion of theGFP-expressing FoxP3⁺ CD4⁺ Treg cells (FIG. 4 ).

P300 inhibition. Inhibition of p300 in mice was performed similarly topreviously described (Liu et al, 2013). p300i (C646; Tocris Bioscience)was dissolved in DMSO and injected i.p. daily (8.9 mg kg⁻¹ d⁻¹, i.p.)for 1 week. Vehicle-treated mice were similarly injected with DMSO.

ATRA treatment. All-trans retinoic acid (ATRA) administration to micewas performed similarly to previously described (Walsh et al, 2014).ATRA (Sigma) was dissolved in DMSO and injected i.p. (8 mg kg⁻¹ d⁻¹)every other day over the course of 1 week. Vehicle-treated mice weresimilarly injected with DMSO.

Soluble Aβ (sAβ) protein isolation and quantification. Tissuehomogenization and sAβ protein extraction was performed as previouslydescribed (Schmidt et al, 2005). Briefly, cerebral brain parenchyma wasdissected and snap-frozen and kept at −80° C. until homogenization.Proteins were sequentially extracted from samples to obtain separatefractions containing proteins of differing solubility. Samples werehomogenized in 10 volumes of ice-cold tissue homogenization buffer,containing 250 mM of sucrose, 20 mM of Tris base, 1 mM ofethylenediaminetetraacetic acid (EDTA), and 1 mM of ethylene glycoltetraacetic acid (pH 7.4), using a ground glass pestle in a Douncehomogenizer. After six strokes, the homogenate was mixed 1:1 with 0.4%diethylamine (DEA) in a 100-mM NaCl solution before an additional sixstrokes, and then centrifuged at 135,000 g at 4° C. for 45 min. Thesupernatant (DEA-soluble fraction containing extracellular and cytosolicproteins) was collected and neutralized with 10% of 0.5Mof Tris-HCl (pH6.8). Aβ₁₋₄₀ and Aβ₁₋₄₂ were individually measured by enzyme-linkedimmunosorbent assay (ELISA) from the soluble fraction using commerciallyavailable kits (Biolegend; # SIG-38954 and # SIG-38956, respectively)according to the manufacturer instructions.

Aβ plaque quantitation. From each brain, 6 μm coronal slices werecollected, and eight sections per mouse, from four differentpre-determined depths throughout the region of interest (dentate gyrusor cerebral cortex) were immunostained. Histogram-based segmentation ofpositively stained pixels was performed using the Image-Pro Plussoftware (Media Cybernetics, Bethesda, Md., USA). The segmentationalgorithm was manually applied to each image, in the dentate gyrus areaor in the cortical layer V, and the percentage of the area occupied bytotal Aβ immunostaining was determined. Plaque numbers were quantifiedfrom the same 6 μm coronal brain slices, and are presented as averagenumber of plaques per brain region. Prior to quantification, slices werecoded to mask the identity of the experimental groups, and plaque burdenwas quantified by an observer blinded to the identity of the groups.

Statistical analysis. The specific tests used to analyze each set ofexperiments are indicated in the figure legends. Data were analyzedusing a two-tailed Student's t test to compare between two groups,one-way ANOVA was used to compare several groups, followed by theNewman-Keuls post-hoc procedure for pairwise comparison of groups afterthe null hypothesis was rejected (P<0.05). Data from behavioral testswere analyzed using two-way repeated-measures ANOVA, and Bonferronipost-hoc procedure was used for follow-up pairwise comparison. Samplesizes were chosen with adequate statistical power based on theliterature and past experience, and mice were allocated to experimentalgroups according to age, gender, and genotype. Investigators wereblinded to the identity of the groups during experiments and outcomeassessment. All inclusion and exclusion criteria were pre-establishedaccording to the IACUC guidelines. Results are presented as means±s.e.m.In the graphs, y-axis error bars represent s.e.m. Statisticalcalculations were performed using the GraphPad Prism software (GraphPadSoftware, San Diego, Calif.).

Example 1. Choroid Plexus (CP) Gateway Activity Along DiseaseProgression in the Mouse Model of AD

We first examined CP activity along disease progression in the 5XFADtransgenic mouse model of AD (AD-Tg); these mice co-express fivemutations associated with familial AD and develop cerebral Aβ pathologyand gliosis as early as 2 months of age (Oakley et al, 2006). We foundthat along the progressive stages of disease pathology, the CP of AD-Tgmice, compared to age-matched wild-type (WT) controls, expressedsignificantly lower levels of leukocyte homing and traffickingdeterminants, including icam1, vcam1, cxcl10, and ccl2 (FIG. 1A), shownto be upregulated by the CP in response to acute CNS damage, and neededfor transepithelial migration of leukocytes (Kunis et al, 2013; Shechteret al, 2013). Immunohistochemical staining for the integrin ligand,ICAM-1, confirmed its reduced expression by the CP epithelium of AD-Tgmice (FIG. 1 b ). In addition, staining for ICAM-1 in human postmortembrains, showed its age-associated reduction in the CP epithelium, inline with our previous observations (Baruch et al, 2014), andquantitative assessment of this effect revealed further decline in ADpatients compared to aged individuals without CNS disease (FIG. 2A).Since the induction of leukocyte trafficking determinants by the CP isdependent on epithelial interferon (IFN)-γ signaling (Kunis et al,2013), we next tested whether the observed effects could reflect loss ofIFN-γ availability at the CP. Examining the CP of 5XFAD AD-Tg mice usingflow cytometry intracellular staining, revealed significantly lowernumbers of IFN-γ-producing cells in this compartment (FIG. 2B), andquantitative real-time PCR (RT-qPCR) analysis confirmed lower mRNAexpression levels of ifn-γ at the CP of AD-Tg mice compared toage-matched WT controls (FIG. 2C).

Example 2. The Functional Relationships Between Treg-Mediated SystemicImmune Suppression, CP Gateway Activity, and AD Pathology

Regulatory T cells (Tregs) play a pivotal role in suppressing systemiceffector immune responses (Sakaguchi et al, 2008). We envisioned thatTreg-mediated systemic immune suppression affects IFN-γ availability atthe CP, and therefore focused on the involvement of Tregs in ADpathology. In line with previous reports of elevated Treg levels andsuppressive activities in AD patients (Rosenkranz et al, 2007; Saresellaet al, 2010; Torres et al, 2013), evaluating Foxp3⁺ Treg frequencies insplenocytes of 5XFAD AD-Tg mice, relative to their age-matched VVTlittermates, revealed their elevated levels along disease progression(FIG. 3A, B). To study the functional relationships betweenTreg-mediated systemic immune suppression, CP gateway activity, and ADpathology, we crossbred 5XFAD AD-Tg mice with Foxp3-diphtheria toxinreceptor (DTR⁺) mice, enabling transient conditional in vivo depletionof Foxp3⁺ Tregs in AD-Tg/DTR⁺ mice by administration of diphtheria toxin(DTx) (FIG. 4A). Transient depletion of Tregs resulted in elevated mRNAexpression of leukocyte trafficking molecules by the CP of AD-Tg/DTR⁺mice relative to DTx-treated AD-Tg/DTR⁻ littermates (FIG. 5A). Analysisof the long-term effect of the transient Treg depletion on the brainparenchyma (3 weeks later), revealed immune cell accumulation in thebrain, including elevated numbers of CD45^(high)/CD11b^(high) myeloidcells, representing infiltrating mo-Mϕ (Shechter et al, 2013), and CD4⁺T cells (FIG. 5B). In addition, the short and transient depletion ofTregs resulted in a marked enrichment of Foxp3⁺ Tregs among the CD4⁺ Tcells that accumulated within the brain, as assessed by flow cytometry(FIG. 5C, D). RT-qPCR analysis of the hippocampus showed increasedexpression of foxp3 and 1110 mRNA (FIG. 5E).

We next examined whether the short-term depletion of Tregs, which wasfollowed by accumulation of immunoregulatory cells in sites of brainpathology, led to a long-term effect on brain function. We observedreduction in hippocampal gliosis (FIG. 5F), and reduced mRNA expressionlevels of pro-inflammatory cytokines, such as it-12p40 and tnf-α (FIG.5G). Moreover, cerebral Aβ plaque burden in the hippocampal dentategyrus, and the cerebral cortex (5^(th) layer), two brain regionsexhibiting robust Aβ plaque pathology in 5XFAD AD-Tg mice(Oakley et al,2006), was reduced (FIG. 6A, B). Evaluating the effect on cognitivefunction, using the Morris water maze (MWM) test, revealed a significantimprovement in spatial learning and memory in AD-Tg/DTR⁺ mice followingthe Treg depletion, relative to DTx-treated AD-Tg/DTR⁻ aged matchedmice, reaching performance similar to that of VVT mice (FIG. 6C-E).Taken together, these data demonstrated that transiently breakingTreg-mediated systemic immune suppression in AD-Tg mice resulted inaccumulation of inflammation-resolving cells, including mo-Mϕ and Tregs,in the brain, and was followed by resolution of the neuroinflammatoryresponse, clearance of Aβ, and reversal of cognitive decline.

Example 3. Weekly Administration of Copolymer-1 Reduces Treg-MediatedSystemic Immune Suppression, Improves CP Gateway Activity, and MitigatesAD Pathology

To further substantiate the causal nature of the inverse relationshipbetween systemic immune suppression, CP function and AD pathology, wenext made use of the immunomodulatory compound, Glatiramer acetate (GA;also known as Copolymer-1, or Copaxone®), which in a weeklyadministration regimen was found to have a therapeutic effect in theAPP/PS1 mouse model of AD (Butovsky et al, 2006); this effect wasfunctionally associated with mo-Mϕ recruitment to cerebral sites ofdisease pathology (Butovsky et al, 2007). Here, we first examinedwhether the CP in APP/PS1 AD-Tg mice, similarly to our observation in5XFAD AD-Tg mice, is also deficient with respect to IFN-γ expressionlevels. We found that in APP/PS1 AD-Tg mice, IFN-γ levels at the CP werereduced relative to age-matched WT controls (FIG. 7A). These resultsencouraged us to test whether the therapeutic effect of weekly-GA inAPP/PS1 mice (Butovsky et al, 2006), could be reproduced in 5XFAD AD-Tgmice, and if so, whether it would affect systemic Tregs, and activationof the CP for mo-Mϕ trafficking. We therefore treated 5XFAD AD-Tg micewith a weekly administration regimen of GA over a period of 4 weeks(henceforth, “weekly-GA”; schematically depicted in FIG. 8A). We foundthat 5XFAD AD-Tg mice treated with weekly-GA, showed reducedneuroinflammation (FIG. 8B-D), and improved cognitive performance, whichlasted up to 2 months after the treatment (FIG. 8E-1 ). Examining byflow cytometry the effect of weekly-GA on systemic immunity and on theCP, we found reduced splenocyte Foxp3⁺ Treg levels (FIG. 9A), and anincrease in IFN-γ-producing cells at the CP of the treated 5XFAD AD-Tgmice, reaching similar levels as those observed in WT controls (FIG.9B). The elevated level of IFN-γ-expressing cells at the CP in theweekly-GA treated mice, was accompanied by upregulated epithelialexpression of leukocyte trafficking molecules (FIG. 9C).

To detect infiltrating mo-Mϕ entry to the CNS, we used 5XFADAD-Tg/CX₃CR1^(GFP/+) bone marrow (BM) chimeric mice (prepared using headprotection), allowing the visualization of circulating (greenfluorescent protein (GFP)⁺ labeled) myeloid cells (Shechter et al, 2009;Shechter et al, 2013). We found increased homing of GFP⁺ mo-Mϕ to the CPand to the adjacent ventricular spaces following weekly-GA treatment, ascompared to vehicle-treated AD-Tg/CX3CR1^(GFP/+) controls (FIG. 9D-E).Immunohistochemistry of the brain parenchyma revealed the presence ofGFP⁺ mo-Mϕ accumulation at sites of cerebral plaque formation (FIG. 9F),and quantification of infiltrating myeloid cells, by flow cytometryanalysis of the hippocampus in AD-Tg non-chimeric mice, showed increasednumbers of CD11b^(high)CD45^(high)-expressing cells (FIG. 9G, H).Together, these results substantiated the functional linkage betweenmo-Mϕ recruitment to sites of AD pathology, reduction of systemic Treglevels and IFN-γ-dependent activation of the CP.

Example 4. Interference with Treg Activity Using a Small MoleculeHistone Acetyltransferase Inhibitor

The findings above, which suggested that Treg-mediated systemic immunesuppression interferes with the ability to fight AD pathology, arereminiscent of the function attributed to Tregs in cancer immunotherapy,in which these cells hinder the ability of the immune system to mount aneffective anti-tumor response (Bos & Rudensky, 2012; Nishikawa &Sakaguchi, 2010). Therefore, we considered that a treatment thatdirectly interferes with Foxp3⁺ Treg cell activity might be advantageousin AD. We tested p300i (C646 (Bowers et al, 2010)), a nonpeptidicinhibitor of p300, a histone acetyltransferase that regulates Tregfunction (Liu et al, 2013); this inhibitor was shown to affect Tregsuppressive activities while leaving protective T effector cellresponses intact (Liu et al, 2013). We found that mice treated withp300i, compared to vehicle (DMSO) treated controls, showed elevatedlevels of systemic IFN-γ-expressing cells in the spleen (FIG. 10A), aswell as in the CP (FIG. 10B). We next treated AD-Tg mice with eitherp300i or vehicle over the course of 1 week, and examined the animals 3weeks later for cerebral Aβ plaque burden. Immunohistochemical analysisrevealed a significant reduction in cerebral AR plaque load in the p300itreated AD-Tg mice (FIG. 10C-E). We also tested whether the effect onplaque pathology following one course of treatment would last beyond the3 weeks, and if so, whether additional courses of treatment wouldcontribute to a long-lasting effect. We therefore compared AD-Tg micethat received a single course of p300i treatment and were examined 2month later, to an age-matched group that received two courses oftreatments during this period, with a 1-month interval in between(schematically depicted in FIG. 10F). We found that the reduction ofcerebral plaque load was evident even two months after a single courseof treatment, but was stronger in mice that received two courses oftreatments with a 1-month interval in between (FIG. 10G). Since impairedsynaptic plasticity and memory in AD is associated with elevatedcerebral levels of soluble Aβ₁₋₄₀/Aβ₁₋₄₂ (sAβ) levels (Shankar et al,2008), we also measured sAβ levels following a single or repeated cyclesof p300i treatment. Again, we found that both one and two courses (withan interval of 1 month in between) were effective in reducing cerebralsAβ, yet this effect was stronger following repeated courses withrespect to the effect on sAβ₁₋₄₂ (FIG. 10H). These results indicatedthat while a single short-term course of treatment is effective,repeated courses of treatments would be advantageous to maintain along-lasting therapeutic effect, similar to our observations followingweekly-GA treatment.

Example 5. Therapeutic Potential of PD-1 Immune Checkpoint Blockade inAlzheimer's Disease

We first tested whether targeting the PD-1 inhibitory pathway couldaffect IFN-γ-associated systemic immunity in 5XFAD AD transgenic (AD-Tg)mice, which co-expresses five mutations associated with familial AD(Oakley et al, 2006). AD-Tg mice at the age of 10 months, a time pointat which cerebral pathology is advanced, were administrated with twointraperitoneal (i.p.) injections of either blocking antibodies directedat PD-1 (anti-PD-1) or IgG control antibodies, on days 1 and 4, and thenexamined on day 7. Flow cytometry analysis revealed that blockade of thePD-1 pathway resulted in elevated frequencies of IFN-γ-producing CD4⁺ Tsplenocytes (FIG. 11A, B).

TABLE 3 GO annotation, related to FIG. 11. FDR GO term DescriptionP-value q-value GO:0034341 response to interferon-gamma 2.13E−142.30E−10 GO:0048002 antigen processing and presentation 3.05E−101.65E−06 of peptide antigen GO:0019886 antigen processing andpresentation 4.11E−10 1.48E−06 of exogenous peptide antigen via MHCclass II GO:0002478 antigen processing and presentation 5.26E−101.42E−06 of exogenous peptide antigen GO:0034097 response to cytokine5.67E−10 1.22E−06 GO:0002504 antigen processing and presentation1.04E−09 1.87E−06 of peptide or polysaccharide antigen via MHC class IIGO:0002495 antigen processing and presentation 1.04E−09 1.60E−06 ofpeptide antigen via MHC class II GO:0019884 antigen processing andpresentation 5.82E−09 7.86E−06 of exogenous antigen GO:0019882 antigenprocessing and presentation 1.43E−07 1.71E−04 GO:0035456 response tointerferon-beta 6.67E−07 7.20E−04 GO:0006955 immune response 1.07E−061.05E−03 GO:0002819 regulation of adaptive immune response 1.92E−061.73E−03 GO:0071345 cellular response to cytokine stimulus 2.21E−061.84E−03 GO:0071346 cellular response to interferon-gamma 2.21E−061.71E−03 Gene ontology terms enriched in the CP of AD-Tg mice treatedwith anti-PD-1, when compared to IgG treated and untreated AD-Tgcontrols. Log 10 values of all RNA sequences of the CP were rankedaccording to their differential expression levels and analyzed.

We next examined whether this systemic immune response affected the CPactivity. Genome wide RNA-sequencing of the CP (Not shown; the fullanalysis will be disclosed in a report by the present inventors havingthe title of Example 5 and it can be obtained from the inventors uponrequest) showed an expression profile associated with response to IFN-γ(FIG. 11D and Table 3), and real-time quantitative PCR (RT-qPCR)verified elevated IFN-γ mRNA levels at the CP, when compared toIgG-treated or untreated AD-Tg controls (FIG. 11C). These findingsconfirmed a systemic, and CP tissue-specific, IFN-γ immune responsefollowing PD-1 blockade, and encouraged us to next test the effect ondisease pathology.

To examine the functional impact of PD-1 blockade on AD pathology, wetreated 10-month old AD-Tg mice with either anti-PD-1 or IgG controlantibodies, and evaluated the effect on spatial learning and memoryperformance, using the radial arm water maze (RAWM) task.

One month following treatment (two i.p. injections with 3-day interval),anti-PD1 treated AD-Tg mice exhibited a significant improvement incognitive function relative to IgG-treated or untreated age-matchedcontrols, reaching cognitive levels similar to that of age-matched WTmice (FIG. 12A). We next tested whether the benefit of PD-1 blockade oncognitive performance in AD-Tg mice would last beyond 1 month, andwhether additional therapeutic sessions would be advantageous. Wetreated AD-Tg mice with anti-PD-1 at the age of 10 months (“1 session”)or at both 10 and 11 months of age (“2 sessions”), and examined theoutcome on cognitive performance at the age of 12 months (schematicallydepicted in FIG. 12B). Control groups included VVT mice, untreated AD-Tgmice, and AD-Tg mice that received two sessions of IgG treatment. Wefound that while a single session of anti-PD-1 administration had abeneficial effect on spatial learning and memory 1 month following thetreatment (FIG. 12A), no significant effect could be detected in micethat received a single session of treatment and were tested 2 monthslater (FIG. 12B). In contrast, AD-Tg mice that received two sessions ofanti-PD-1, at a 1-month interval, displayed cognitive performancesimilar to that of WT mice, at the end of the 2-month timeframe (FIG.12B).

We examined whether PD-1 blockade affected AD pathology as manifested bycerebral AR plaque load and gliosis. Brains of AD-Tg mice that receivedanti-PD-1 or IgG in either one or two sessions were examined byimmunohistochemistry for Aβ and glial fibrillary acid protein (GFAP). Wefound that cerebral Aβ plaque burden was reduced in the hippocampaldentate gyrus (FIG. 13A, B), and the cerebral cortex (5th layer) (FIG.13A, C), two brain regions exhibiting robust Aβ plaque pathology in5XFAD mice (Oakley et al, 2006). The effect on Aβ clearance was evidentfollowing a single session of anti-PD-1 administration, and was morerobust following two sessions. Quantitative analysis of GFAPimmunostaining showed reduced hippocampal astrogliosis in both AD-Tgmice treated with 1 session, and those treated with 2 sessions of PD-1blockade, relative to IgG-treated controls (FIG. 13A, D).

To investigate the effect of dosage and frequency of administration,female 5XFAD AD transgenic mice (average cohorts age of 6 months) weretreated with either anti-PD-1-specific antibody (IgG2a anti-mouse PD-1or IgG control (Rat IgG2a). Anti-PD-1-treated mice received either 1injection of 500 ug of antibody on day 1 of the experiment, or twoinjections of 250 ug with a 3-day interval between injections. Agedmatched wild-type (VVT) mice were used as additional control group.Treatment effect on spatial learning and memory performance ofanti-PD-1-treated 5XFAD mice—one injection (n=7) or two injections(n=11), IgG2a-treated 5XFAD mice (n=10), and WT (n=14) controls wasevaluated using the radial arm water maze (RAWM) task at the age of 7months (FIG. 14 ). Black arrows indicate time points of treatment, andillustrations indicate time points of cognitive testing.Repeated-measures were analyzed using two-way ANOVA and Dunnettpost-test. Error bars represent mean±s.e.m.; *P<0.05, **P<0.01,***P<0.001, anti-PD-1-treated (1 injection) versus IgG-treated controls.One month following treatment (two i.p. injections with 3-day interval),anti-PD1 treated AD-Tg mice exhibited a significant improvement incognitive function relative to IgG-treated or untreated age-matchedcontrols, reaching cognitive levels similar to that of age-matched WTmice (FIG. 14 ).

Finally, male 5XFAD AD transgenic mice were treated in a repeatedtreatment session, once a month, with either anti-PD-1-specific antibody(IgG2a anti-mouse PD-1) or IgG control (Rat IgG2a). The first injectionwas at the age of 3 months, the second at the age of 4 months, and thethird at the age of 5 months. Dosage is indicated in the scheme of theexperimental design (FIG. 15A). Aged matched wild-type (VVT) mice wereused as additional control group. Treatment effect on spatial learningand memory performance was evaluated using the radial arm water maze(RAWM) task, at two different time points—the age of 5 months (FIG.15B), and the age of 6 months (FIG. 15C). Black arrows indicate timepoints of treatment, and illustrations indicate time points of cognitivetesting. RAWM performance of anti-PD-1-treated 5XFAD mice (n=7),IgG2a-treated 5XFAD mice (n=9), and VVT (n=8) controls.Repeated-measures were analyzed using two-way ANOVA and Dunnettpost-test. Error bars represent mean±s.e.m.; *P<0.05, **P<0.01,***P<0.001, anti-PD-1-treated versus IgG-treated controls. Thesefindings demonstrate that repeated sessions of treatment with PD-1blockade, could not only reverse disease progression when given to 5XFADmice at advanced stages of disease, but also delay disease onset whenthe treatment commences at an early age, prior to cognitive decline(FIGS. 15B-C).

Example 6. Therapeutic Potential of TIM-3 Immune Checkpoint Blockade inAlzheimer's Disease

To examine the functional impact of TIM-3 blockade on AD pathology, wetreated 6-month old female 5XFAD AD-Tg mice with eitheranti-TIM-3-specific antibody (anti-mouse TIM-3) or IgG control (RatIgG2a antibody). Dosage is indicated in the scheme of the experimentaldesign (FIG. 16 ). The treatment was consistent of two i.p. injectionsof the antibody, 250 μg each, with a 3-day interval between injections.Aged matched wild-type (VVT) mice were used as additional control group.Treatment effect on spatial learning and memory performance ofanti-TIM-3-treated 5XFAD mice (n=9), of IgG-treated (n=6) 5XFAD mice andVVT (n=7) controls was evaluated using the radial arm water maze (RAWM)task at the age of 7 months (FIG. 16 ). Black arrows indicate timepoints of treatment, and illustrations indicate time points of cognitivetesting. Repeated-measures were analyzed using two-way ANOVA and Dunnettpost-test. Error bars represent mean±s.e.m.; *P<0.05, **P<0.01,***P<0.001, anti-TIM-3-treated versus IgG-treated controls. One monthfollowing treatment, anti-TIM-3-treated AD-Tg mice exhibited asignificant improvement in cognitive performance relative to IgG-treated5XFAD mice, or aged-matched VVT controls (FIG. 16 ).

Example 7. Therapeutic Potential of PD-L1 Immune Checkpoint Blockade inAlzheimer's Disease, and Comparison to Anti-PD-1 Treatment

To examine the functional impact of PD-L1 immune checkpoint blockade onAD pathology, and to compare it to the effect of the same dose andregimen of anti-PD-1 blockade, we treated female 5XFAD AD transgenicmice (average cohorts age of 6 months) with either anti-PD-1-specificantibody (IgG2a rat anti-mouse PD-1), anti-PD-L1-specific antibody(IgG2b anti-mouse PD-L1), or IgG control (either Rat IgG2a or IgG2b).Dosage is indicated in the scheme of the experimental design (FIG. 17A).Aged matched wild-type (VVT) mice were used as additional control group.Treatment effect on spatial learning and memory performance ofanti-PD-1-treated 5XFAD mice (n=9), anti-PD-L1-treated 5XFAD mice (n=7),IgG2a-treated 5XFAD mice (n=6), IgG2b-treated 5XFAD mice (n=7) and VVT(n=16) controls was evaluated using the radial arm water maze (RAWM)task at the age of 7 months (FIG. 17A). Black arrows indicate timepoints of treatment, and illustrations indicate time points of cognitivetesting. Repeated-measures were analyzed using one-way ANOVA and Dunnettpost-test. Error bars represent mean±s.e.m.; *P<0.05, **P<0.01,***P<0.001, anti-PD-1-treated versus IgG-treated controls. # P<0.05, ##P<0.01, ### P<0.001, anti-PD-L1-treated versus IgG-treated controls. Onemonth following treatment, anti-PD-L1 and anti-PD-1-treated AD-Tg micewere examined for their cognitive performance and were compared toIgG-treated AD-Tg mice (negative control). Wild type mice were alsoassessed cognitively in this experiment, and were used as a positivecontrol for cognitive performance. Both anti-PD-L1 and anti-PD-1-treatedAD-Tg mice exhibited a similar and significant improvement in cognitiveperformance relative to IgG-treated 5XFAD mice (FIG. 17A).

Following the RAWM test, mice were sacrificed, and their brains werecollected for fixation and histology/immunohisctocimical analysis. Meanplaque area and numbers were quantified (in 6-μm brain slices) in the5th layer of the cerebral cortex, showing plaque load reduction in bothtreatment group (anti-PD-1 and anti-PD-L1). This analysis showed thatboth anti-PD-1 and anti-PD-L1 treated animals showed a statisticallysignificant decrease in Aβ-plaque formation in the cortex (FIG. 17B)

PD-L1 is expressed by activated immune cells such as T cells, B cells,macrophages, dendritic cells and microglia, as well as by non-immunecells such as endothelial and epithelial cells. Thus, we envisioned thatthe expression of PD-L1 by the CP epithelium might contribute to thedown regulation of the trafficking of leukocytes to the CNS, bydampening the activity of IFN-γ producing T-cells which express PD-1upon communication with PD-L1-expressing epithelial cells within the CP.Immunohistochemical analysis shows that in aged mice the CP epitheliumexpressed significantly higher levels of PD-L1, compared to young mice(FIG. 18 ).

Example 8. Therapeutic Potential of Immune Checkpoint Blockade inAlzheimer's Disease

To teste whether blockade of immune checkpoints could attenuate ADpathology, AD-Tg mice are treated at ages between 6 to 10-month old withone of the following anti-checkpoint antibodies: anti-ICOS, anti-B7RP1,anti-VISTA, anti-CD40, anti-CD40L, anti-CD80, anti-CD86, anti-B7-H3,anti-B7-H4, B7-H7, anti-BTLA, anti-HVEM, anti-CD137, anti-CD137L,anti-OX40L, anti-CD-27, anti-CD70, anti-STING, anti-TIGIT antibody oranti-GITR antibody. Some mice are treated with anti-PD-1 antibody aspositive control, IgG control as negative control or combinations ofanti-PD1 and one of the other anti-checkpoint antibodies mentionedabove. Treatment effect on spatial learning and memory performance,using the radial arm water maze (RAWM) task, Aβ plaque burden byimmunohistochemistry for Aβ and hippocampal astrogliosis byimmunohistochemistry for glial fibrillary acid protein (GFAP) will bemeasured one month following treatment.

It is expected that the mice treated with the antibodies displaysignificant cognitive improvement in comparison to IgG-treated anduntreated AD-Tg mice as well as a significant reduction of cerebralplaque load.

Example 9. Therapeutic Potential of PD-1 in Combination with CTLA-4Immune Checkpoint Blockade in Alzheimer's Disease

At 10 months of age, 5XFAD Alzheimer's′ disease (AD) transgenic (Tg)mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA-4(InVivoMAb anti-mCD152; # BE0131; Bioxcell Lifesciences Pvt. LTD.) orcontrol IgG (IgG2a, # BE0089 or Polyclonal Syrian Hamster IgG, # BE0087;Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1 and day 4 of theexperiment, and are examined 3 weeks after for their cognitiveperformance by radial arm water maze (RAWM) spatial learning and memorytask, as described above.

Some mice receive an additional treatment session with an intervalsession of 3 weeks. Control groups are either treated with IgG oruntreated, and all groups of mice are tested for their cognitiveperformance 3 weeks later.

It is expected that the mice treated with the combination of antibodiesdisplay significant cognitive improvement in comparison to IgG-treatedand untreated AD-Tg mice as well as a significant reduction of cerebralplaque load.

Example 10. Therapeutic Potential of Immune Checkpoint Blockade Approachin PTSD Pathology

Severely stressful conditions or chronic stress can lead toposttraumatic stress disorder (PTSD) and depression. We adopted aphysiological PTSD-like animal model in which the mice exhibithypervigilant behavior, impaired attention, increased risk assessment,and poor sleep (Lebow et al, 2012). In this experimental model of PTSDinduction, mice are habituated for 10 days to a reverse day/night cycle,inflicted with two episodes of electrical shocks (the trauma and thetrigger), referred to as a “PTSD induction”, and evaluated at differenttime points subsequent to trauma. Following the traumatic event mice areinjected with said compound which blocks immune checkpoints. The miceare treated according to one of the following regimens:

The mice are treated with one of the following anti-checkpointantibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L,anti-CD80, anti-CD86, anti-B7-H3, anti-B7-H4, B7-H7, anti-BTLA,anti-HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70,anti-STING, anti-GITR or anti-TIGIT antibody alone or in combinationwith an anti-CTLA-4 antibody. Some mice are treated with anti-PD-1antibody as positive control, IgG control as negative control orcombinations of anti-PD1 and one of the other anti-checkpoint antibodiesmentioned above.

Some mice receive an additional treatment session with an appropriateinterval session.

It is expected that mice that receive the treatment do not displayanxiety behavior associated with PTSD in this experimental model, asassessed by time spent exploring and risk assessing in dark/light mazeor the other behavioral tasks described in (Lebow et al, 2012).

Example 11. Therapeutic Potential of Immune Checkpoint Blockade Approachin Parkinson's Disease Pathology

Parkinson disease (PD) transgenic (Tg) mice or the MPTP-induced mousemodels of PD are used in these experiment. The mice are treated at theprogressive stages of disease according to one of the followingregimens:

PD-Tg mice are treated with one of the following anti-checkpointantibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L,anti-CD80, anti-CD86, anti-B7-H3, anti-B7-H4, B7-H7, anti-BTLA,anti-HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70,anti-STING, anti-GITR or anti-TIGIT antibody alone or in combinationwith an anti-CTLA-4 antibody. Some mice are treated with anti-PD-1antibody as positive control, IgG control as negative control orcombinations of anti-PD1 and one of the other anti-checkpoint antibodiesmentioned above.

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod.

It is expected that PD-Tg mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. PD-Tg mice whichreceive two courses of therapy, and examined after an appropriateinterval session are expected to show a long-lasting therapeutic effect.To maintain this therapeutic effect mice are subjected to an activesession of treatment with an appropriate interval session ofnon-treatment between each treatment session.

Example 12. Therapeutic Potential of PD-1 in Combination with CTLA-4Immune Checkpoint Blockade in Huntington's Disease Pathology

The model used in these experiments may be the Huntington's disease (HD)R6/2 transgenic mice (Tg) test system. R6/2 transgenic mice over expressthe mutated human huntingtin gene that includes the insertion ofmultiple CAG repeats mice at the progressive stages of disease. Thesemice show progressive behavioral-motor deficits starting as early as 5-6weeks of age, and leading to premature death at 10-13 weeks. Thesymptoms include low body weight, clasping, tremor and convulsions.

The mice are treated according to one of the following regimens whenthey are 45 days old:

The mice are treated with one of the following anti-checkpointantibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L,anti-CD80, anti-CD86, anti-B7-H3, anti-B7-H4, B7-H7, anti-BTLA,anti-HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70,anti-STING, anti-GITR or anti-TIGIT antibody alone or in combinationwith an anti-CTLA-4 antibody. Some mice are treated with anti-PD-1antibody as positive control, IgG control as negative control orcombinations of anti-PD1 and one of the other anti-checkpoint antibodiesmentioned above.

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod.

It is expected that HD-Tg mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. HD-Tg mice whichreceive which receive two courses of therapy, and examined after anappropriate interval session are expected to show a long-lastingtherapeutic effect. To maintain this therapeutic effect mice aresubjected to an active session of treatment with an appropriate intervalsession of non-treatment between each treatment session.

Example 13. Therapeutic Potential of Immune Checkpoint Blockade Approachin Amyotrophic Lateral Sclerosis Pathology

The model used in this experiment may be the transgenic miceoverexpressing the defective human mutant SOD1 allele containing theGly93→Ala (G93A) gene (B6SJL-TgN (SOD1-G93A)1Gur (herein “ALS mice”).This model develop motor neuron disease and thus constitute an acceptedanimal model for testing ALS.

The mice are treated according to one of the following regimens whenthey are 75 days old:

The mice are treated with one of the following anti-checkpointantibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L,anti-CD80, anti-CD86, anti-B7-H3, anti-B7-H4, B7-H7, anti-BTLA,anti-HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70,anti-STING, anti-GITR or anti-TIGIT antibody alone or in combinationwith an anti-CTLA-4 antibody. Some mice are treated with anti-PD-1antibody as positive control, IgG control as negative control orcombinations of anti-PD1 and one of the other anti-checkpoint antibodiesmentioned above.

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod, or mice are allowed to grasp and hold onto a vertical wire(2 mm in diameter) with a small loop at the lower end. A vertical wireallows mice to use both fore- and hindlimbs to grab onto the wire. Thewire is maintained in a vertically oriented circular motion (the circleradius was 10 cm) at 24 rpm. The time that the mouse is able to hangonto the wire is recorded with a timer.

It is expected that ALS mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. ALS mice whichreceive which receive two courses of therapy, and examined after anappropriate interval session are expected to show a long-lastingtherapeutic effect. To maintain this therapeutic effect mice aresubjected to an active session of treatment with an appropriate intervalsession of non-treatment between each treatment session.

Example 14. Dose Effect Experiments to Determine Minimal and MaximalDose Range and Experiments to Determine Treatment Regimen and its LongLasting Therapeutic Effect

We already showed that a single treatment session utilizing PD-1blockade leads to a significant reduction in plaque burden and improvedcognitive function that lasts for at least 2 months after the treatment,the last time point that was tested. Here we describe a dose responsestudy using two additional dosages administered to 5XFAD AD transgenicmice. The readout will be amyloid plaque burden at one, two and threemonths post administration. The study groups will include 1) untreated5XFAD mice; 2) 5XFAD mice which receive 1 injection of 500 μg controlanti-PD-1 (RMP1-14; # BE0146; Bioxcell Lifesciences Pvt. LTD.); 3) 5XFADmice which receive 1 injection of 250 μg control anti-PD-1 (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.); 4) 5XFAD mice which receive 1injection of 100 μg control anti-PD-1 (RMP1-14; # BE0146; BioxcellLifesciences Pvt. LTD.); and 5) 5XFAD mice which receive 1 injection of500 μg control IgG (IgG2a; # BE0089; Bioxcell Lifesciences Pvt. LTD.).All of the mice are treated at the start of the experiment, and fromeach group mice are sacrificed and their brains are examined atintervals of 1 month, 2 months, and 3 months following the start of thetreatment.

It is expected that the mice treated with the anti-PD-1 antibodiesdisplay significant reduction in cerebral amyloid beta plaque load incomparison to untreated AD-Tg mice or to control IgG-treated mice.

An additional treatment session with anti-PD-1, a month after theinitial treatment, was found by us to maintain the effect on cognitiveperformance improvement in 5XFAD AD-Tg mice (Example 5). These findingssuggest that for long-term efficacy, repeated treatment sessions areneeded. Here we describe a study using repeated injections formaintaining the long-lasting effect of the therapy.

5XFAD AD-Tg mice are injected with the drug at a dosage that will bedetermined according to the previous study results. Mice will beinjected and their cognitive performance is monitored using the radialarm water maze learning and memory task during and after the studyperiod. Histological examination of the brain for amyloid plaque burdenis also performed.

Different groups of mice are injected repeatedly with single injections(or double injections 3 days apart as described in Example 5) with 2, 3or 4 weeks intervals of non-treatment (Table 4). The mice are monitoredas described above at one, two or three months after the initialtreatment.

TABLE 4 Frequency of administration and timing of tests Frequency Week 01 2 3 4 5 6 7 8 9 10 11 12 2 x x x x x 3 x x x x 4 x x x Test T T T

Example 15. Systemic Administration of Anti-PD-1 Monoclonal Antibody inRCS Rats Attenuates Retinal Degeneration

The aim of this experiment was to determine whether systemicadministration of an anti-PD1 antibody attenuates the degeneration ofthe outer nuclear layer in an animal model of a retinal degenerationdisease.

RCS rats, an accepted animal model of dry AMD and Retinitis Pigmentosaand other retinal degenerative diseases and conditions, carry a deletionmutation in the gene encoding the MerTK protein which leads to retinaldegeneration and complete loss of sight by the age of three months. Asignificant and rapid deterioration in the thickness of the retinalouter nuclear layer (ONL) is observed starting at the age of 4 weeks.Preservation of ONL thickness in this model is thus considered in thescientific literature to be directly correlated to preservation ofsight.

RCS rats were injected intraperitoneally (IP) at the age of 4 weeks witheither anti-PD1 monoclonal antibodies (total of 760 μg per animal; n=10)or suitable IgG control (IgG2a) at the same concentration (n=10). Anadditional group of RCS rats were left untreated (n=4). 2 weekspost-treatment, at the age of 6 weeks, the animals were sacrificed,their eyes excised and the thickness of the retinal ONL layer in eacheye was determined through histological analysis using H&E stain.Treatment efficacy was determined by measuring the ONL thicknessthroughout the entire retinal length and horizontally plotting the datato generate a map that allows identification of treatment effects in anyarea of the retina.

Analyzing the mean ONL thickness of all treated animals where eachindividual eye served as an independent data set, showed that at the ageof 6 weeks (2 weeks after treatment), the ONL at the central area of theretina in close proximity to the optic nerve head was significantlythicker in anti-PD1 treated animals compared to both IgG-treated anduntreated controls (FIG. 19A).

To evaluate the magnitude of the effect in treatment-responsive animalsonly, a threshold value for defining an animal as a ‘positive responsiveanimal’ was set as a value equals to 2 standard error values above themean thickness value in the central retina of the IgG control group.Based on this set threshold value, 13 of the 20 analyzed eyes (65%) inthe anti-PD1 group were characterized as positive-responders. Incomparison, only 1 of 20 (5%) analyzed eye was characterized as apositive-responder in the IgG treatment group; a significant difference(Chi-square=15.82, P=0.00007). Looking only at the 13 anti-PD1positive-responsive eyes of this treatment group showed that thethickness at the central retina area was 1.5-2 times double of that ofthe control eyes. Moreover, an even wider area of the central retina wassignificantly thicker compared to the control groups (FIG. 19B).Interestingly, although the antibody was administered systemically andis expected to reach and act on both eyes at a similar level,differences in responsiveness to the treatment between the two eyes ofindividual animals were observed in four of the anti-PD-1 treatedanimals.

Example 16. Local Administration of Anti-PD-1 Monoclonal AntibodyDirectly into the Vitreous in RCS Rats Attenuates Retinal Degeneration

The aim of this experiment was to determine whether local administrationof an anti-PD1 antibody directly into the vitreous of the eye attenuatesthe degeneration of the outer nuclear layer in an animal model of aretinal degeneration disease.

RCS rats at the age of 4 weeks were injected with either anti-PD1monoclonal antibodies (n=6) or suitable IgG control (50 μg per animal).Injection was performed directly into the vitreous of a single eye ofeach animal while the contralateral eye was left untreated. 2 weekspost-treatment, at the age of 6 weeks, the animals were sacrificed,their eyes excised and the thickness of the retinal ONL layer in eacheye was determined through histological analysis using H&E stain.Treatment efficacy was determined by measuring the ONL thicknessthroughout the entire retinal length and horizontally plotting the datato generate a map that allows identification of treatment effects in anyarea of the retina.

Analyzing the mean ONL thickness of the animals 2 weeks after treatmentshowed that in the anti-PD1-treated group, the ONL was significantlythicker in several sections of the retina in the treated eye compared tothe untreated contralateral eye (FIG. 20A). No such effect was found inthe IgG-treated animals (FIG. 20B).

Example 17. Local PD-1 Blockade Through Intravitreal Injection of PD-1or PD-L1 Monoclonal Antibodies Attenuates Retinal Degeneration

Anti-PD-L1 monoclonal antibodies or anti-PD-1 monoclonal antibodies, acombination of Anti-PD-L1 and anti-PD-1 monoclonal antibodies orsuitable IgG fragments without the antigen-specific variable region,will be injected directly into the vitreous of RCS rats at 4 weeks ofage. Throughout the 8 following weeks, assessment of visual function ofthe animals in response to visual stimuli will be taken. Importantly,only a single eye of each animal will receive the treatment while thecontra-lateral eye will be left untreated and will serve as anadditional control. In addition, throughout the experiment, designatedgroups of RCS rats from each treatment group will be subjected toanalysis aiming at quantifying and qualifying the treatment effect onthe retina in term of neuronal survival and local immune response, aswell as the effect on the retinal epithelial cells in terms of theirexpression of leukocyte trafficking molecules and immune checkpointligands. Local blockade of the PD-1/PD-L1 pathway through intravitrealinjection is expected to result in attenuation of retinal degeneration,immune modulation and preservation of visual function.

Example 18. PD-1 Blockade Beneficial Effect on Memory Performance in theDM-Htau Mouse Model of Alzheimer's Disease

Male mice expressing the human-tau (htau) gene with two mutations(K257T/P301S; double mutant, DM (Rosenmann et al., 2008) were treated atthe age of 7 months with either anti-PD-1-specific antibody (IgG2a ratanti-mouse PD-1; n=5), or IgG control (Rat IgG2a; n=5), and were testedby T-maze cognitive task a month after. Aged matched wild-type (WT; n=5)non-transgenic littermates mice were used as additional control group.The experiment was performed in a blinded fashion to the identity of thetreatment groups—both during T-maze testing, and during quantificationof time spent in each arm of the T-maze. Repeated-measures were analyzedusing one-way ANOVA and Tukey's post-test. Error bars representmean±s.e.m.; *P<0.05. The results show that systemic blockade of thePD-1/PD-L1 pathway triggers a protective immune activity which mitigatesthe pathological events in the brain of AD mice also in the absence ofamyloid beta related pathology. The DM-htau model mimics the humanpathology of abnormal human tau protein in the brain, which appears inother diseases (in addition to Alzheimer's disease) collectively calledtauopathies. The list of tauopathies include Pick's disease, progressivesupranuclear palsy, corticobasal degeneration, argyrophilic graindisease, primary age-related tauopathy, formerly called also asneurofibrillary tangleonly dementia, and a recently characterized entitycalled globular glial tauopathy (FIG. 21 ).

Example 19. PD-1 Blockade Enhances Hippocampal Neurogenesis in 5XFADMice

Doublecortin (DCX) is a microtubule-associated protein expressed byneuronal precursor cells and immature neurons. In the adult neuronaltissue DCX is used as a marker for neurogenesis since it is expressedalmost solely by developing neurons. Female 5XFAD mice (average cohortsaged 6 months) were treated with either anti-PD-1-specific antibody(IgG2a anti-mouse PD-1; n=17), or IgG control (Rat IgG2a; n=7), and weresacrificed a month after. Aged matched wild-type (WT; n=9) mice wereused as additional control group. Parasagittal brain sections fromrepresentative animals were prepared and the granular layer of thedentate gyrus was marked (FIG. 22A). Brain sections were immunostainedfor neuronal marker-NeuN (in green), DCX (in red), and hoechst nuclearstaining (in blue). DCX+ cells were quantified in a double-blindedmanner from 6 m thick brain slices (FIG. 22B). Repeated measures wereanalyzed using one-way ANOVA and Dunnett post-test. Error bars representmean±s.e.m.; *P<0.05, **P<0.01, ***P<0.001. The results show thatsystemic blockade of PD-1/PD-L1 pathway triggers a protective immuneactivity which modulates the brain environment to support hippocampalneurogenesis, an effect on pathology which was repeatedly correlatedbefore to a beneficial effect on behavior deficits and cognitivedeficits.

Example 20. PD-1 Blockade Enhances Hippocampal Synaptic Plasticity in5XFAD Mice

Vesicular glutamate transporters 1 (VGLUT1), expressed by glutamatergicneurons, mediate glutamate uptake into synaptic vesicles and was shownto contribute to hippocampal synaptic plasticity andhippocampus-dependent spatial learning. Female 5XFAD mice (averagecohorts aged 6 months) were treated with either anti-PD-1-specificantibody (IgG2a anti-mouse PD-1; n=17), or IgG control (Rat IgG2a; n=7),and were sacrificed a month after. Aged matched wild-type (WT; n=9) micewere used as additional control group. Parasagittal brain sections fromrepresentative animals were prepared and the Subiculum region was marked(FIG. 23A). Brain sections were immunostained for VgluT1. Florescenceintensity was quantified in a double-blinded manner using the ImageJsoftware from 6 m thick brain slices (FIG. 23B). Repeated measures wereanalyzed using one-way ANOVA and Dunnett post-test). Error barsrepresent mean±s.e.m.; *P<0.05, **P<0.01, ***P<0.001. The results showthat systemic blockade of PD-1/PD-L1 pathway triggers a protectiveimmune activity in the periphery and which modulates the brainenvironment to support synaptic plasticity and preserve cognitivefunction.

Example 21. PD-1 Blockade Reduces Neuronal Loss in the Subiculum of5XFAD Mice

The 5XFAD transgenic mouse model is one of the few amyloid animal modelsthat exhibits significant neuron loss, similar to AD progression inhuman patients. Neuronal loss in 5XFAD mice was characterized in theSubiculum and cortical Layer 5. Female 5XFAD mice (average cohorts aged6 months) were treated with either anti-PD-1-specific antibody (IgG2aanti-mouse PD-1; n=17), or IgG control (Rat IgG2a; n=7), and weresacrificed a month after. Aged matched wild-type (WT; n=9) mice wereused as additional control group. Parasagittal brain sections fromrepresentative animals were prepared and the Subiculum region was marked(FIG. 24A). Brain sections were immunostained for NeuN, labeling neurons(in green). Subiculum neurons were quantified in a double-blinded mannerusing the ImageJ software from 6 m thick brain slices (FIG. 24B).Repeated measures were analyzed using one-way ANOVA and Dunnettpost-test). Error bars represent mean±s.e.m.; *P<0.05, **P<0.01,***P<0.001. The results show that systemic blockade of PD-1/PD-L1pathway triggers a protective immune activity which modulates the brainenvironment to become permissive for support neuronal survival andrescue, eventually contributing to better cognitive performance.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particular compound,composition, article, apparatus, methodology, protocol, and/or reagent,etc., described herein, unless expressly stated as such. In addition,those of ordinary skill in the art will recognize that certain changes,modifications, permutations, alterations, additions, subtractions andsub-combinations thereof can be made in accordance with the teachingsherein without departing from the spirit of the present specification.It is therefore intended that the following appended claims and claimshereafter introduced are interpreted to include all such changes,modifications, permutations, alterations, additions, subtractions andsub-combinations as are within their true spirit and scope.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. For instance, as massspectrometry instruments can vary slightly in determining the mass of agiven analyte, the term “about” in the context of the mass of an ion orthe mass/charge ratio of an ion refers to +/−0.50 atomic mass unit. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalindication should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Use of the terms “may” or “can” in reference to an embodiment or aspectof an embodiment also carries with it the alternative meaning of “maynot” or “cannot.” As such, if the present specification discloses thatan embodiment or an aspect of an embodiment may be or can be included aspart of the inventive subject matter, then the negative limitation orexclusionary proviso is also explicitly meant, meaning that anembodiment or an aspect of an embodiment may not be or cannot beincluded as part of the inventive subject matter. In a similar manner,use of the term “optionally” in reference to an embodiment or aspect ofan embodiment means that such embodiment or aspect of the embodiment maybe included as part of the inventive subject matter or may not beincluded as part of the inventive subject matter. Whether such anegative limitation or exclusionary proviso applies will be based onwhether the negative limitation or exclusionary proviso is recited inthe claimed subject matter.

Notwithstanding that the numerical ranges and values setting forth thebroad scope of the invention are approximations, the numerical rangesand values set forth in the specific examples are reported as preciselyas possible. Any numerical range or value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Recitation of numerical rangesof values herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar references used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Further, ordinal indicators—such as “first,” “second,” “third,”etc. —for identified elements are used to distinguish between theelements, and do not indicate or imply a required or limited number ofsuch elements, and do not indicate a particular position or order ofsuch elements unless otherwise specifically stated. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the presentinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the present specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

When used in the claims, whether as filed or added per amendment, theopen-ended transitional term “comprising” (and equivalent open-endedtransitional phrases thereof like including, containing and having)encompasses all the expressly recited elements, limitations, stepsand/or features alone or in combination with unrecited subject matter;the named elements, limitations and/or features are essential, but otherunnamed elements, limitations and/or features may be added and stillform a construct within the scope of the claim. Specific embodimentsdisclosed herein may be further limited in the claims using theclosed-ended transitional phrases “consisting of” or “consistingessentially of” in lieu of or as an amended for “comprising.” When usedin the claims, whether as filed or added per amendment, the closed-endedtransitional phrase “consisting of” excludes any element, limitation,step, or feature not expressly recited in the claims. The closed-endedtransitional phrase “consisting essentially of” limits the scope of aclaim to the expressly recited elements, limitations, steps and/orfeatures and any other elements, limitations, steps and/or features thatdo not materially affect the basic and novel characteristic(s) of theclaimed subject matter. Thus, the meaning of the open-ended transitionalphrase “comprising” is being defined as encompassing all thespecifically recited elements, limitations, steps and/or features aswell as any optional, additional unspecified ones. The meaning of theclosed-ended transitional phrase “consisting of” is being defined asonly including those elements, limitations, steps and/or featuresspecifically recited in the claim whereas the meaning of theclosed-ended transitional phrase “consisting essentially of” is beingdefined as only including those elements, limitations, steps and/orfeatures specifically recited in the claim and those elements,limitations, steps and/or features that do not materially affect thebasic and novel characteristic(s) of the claimed subject matter.Therefore, the open-ended transitional phrase “comprising” (andequivalent open-ended transitional phrases thereof) includes within itsmeaning, as a limiting case, claimed subject matter specified by theclosed-ended transitional phrases “consisting of” or “consistingessentially of.” As such embodiments described herein or so claimed withthe phrase “comprising” are expressly or inherently unambiguouslydescribed, enabled and supported herein for the phrases “consistingessentially of” and “consisting of.”

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

The invention claimed is:
 1. A method of treating a retinal degenerationdisorder in an individual in need thereof, the method comprisingadministering to the individual a composition comprising an anti-PD-1antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, or anycombination thereof.
 2. The method according to claim 1, wherein theadministration of the composition is a single administration.
 3. Themethod according to claim 1, wherein the administration of thecomposition is a repeated administration.
 4. The method according toclaim 1, wherein the administration is a local administration.
 5. Themethod according to claim 4, wherein the local administration is to atleast one eye of the individual.
 6. The method according to claim 5,wherein the local administration is an intravitreal administration. 7.The method according to claim 1, wherein the administration is asystemic administration.
 8. The method according to claim 1, wherein thecomposition is administered by a dosage regime comprising at least twocourses of therapy, each course of therapy comprising in sequence atreatment session where the composition is administered to theindividual followed by a non-treatment period where the composition isnot administered to the individual.
 9. The method according to claim 8,wherein the non-treatment period is longer than the treatment session.10. The method according to claim 8, wherein the administration of thecomposition is a single administration.
 11. The method according toclaim 8, wherein the administration of the composition is a repeatedadministration.
 12. The method according to claim 11, wherein thenon-treatment period is longer than the period between repeatedadministrations during the treatment session.
 13. The method accordingto claim 1, wherein the anti-PD-1 antibody is a neutralizing anti-PD-1antibody, the anti-PD-L1 antibody is a neutralizing anti-PD-L1 antibodyand/or the anti-TIM-3 antibody is a neutralizing anti-TIM-3 antibody.14. The method according to claim 13, wherein the anti-PD-1 antibody isa human neutralizing anti-PD-1 antibody or a humanized, neutralizinganti-PD-1 antibody.
 15. The method according to claim 13, wherein theanti-PD-L1 antibody is a human neutralizing anti-PD-L1 antibody or ahumanized, neutralizing anti-PD-L1 antibody.
 16. The method according toclaim 13, wherein the anti-TIM-3 antibody is a human neutralizinganti-TIM-3 antibody or a humanized, neutralizing anti-TIM-3 antibody.17. The method according to claim 1, wherein the retinal degenerationdisorder is wet Age-Related Macular Degeneration, dry Age-RelatedMacular Degeneration, Retinitis Pigmentosa, Choroideremia, Cone-RodRetinal Dystrophy, Gyrate Atrophy, Juvenile Retinoschisis, VitelliformMacular Dystrophy (Best's Disease), Abetalipoproteinemia(Bassen-Kornzweig Disease), Bardet-Biedl Syndrome, Blue ConeMonochromatism Disease, Dominant Drusen, Goldman-Favre VitreoretinalDystrophy (Enhanced S-cone Syndrome), Kearns-Sayre Syndrome,Laurence-Moon Syndrome, Leber's Congenital Amaurosis, Leber's Refsumdisease, Oguchi Disease, Peripapillary (pericentral) ChoroidalDystrophy, Pigment Pattern Dystrophy, Sorsby Macular Dystrophy,Stargardt's Disease, Stickler's Syndrome, Usher Syndrome or Wagner'sVitreoretinal Dystrophy.